WO2006096497A2 - Compositions contenant des molecules conjuguees structurellement stables - Google Patents

Compositions contenant des molecules conjuguees structurellement stables Download PDF

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WO2006096497A2
WO2006096497A2 PCT/US2006/007571 US2006007571W WO2006096497A2 WO 2006096497 A2 WO2006096497 A2 WO 2006096497A2 US 2006007571 W US2006007571 W US 2006007571W WO 2006096497 A2 WO2006096497 A2 WO 2006096497A2
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composition
antigen
iss
conjugate
aic
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PCT/US2006/007571
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English (en)
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WO2006096497A3 (fr
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Stephen F. Tuck
Roberto Rodriguez
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Dynavax Technologies Corporation
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Priority to EP06736828A priority Critical patent/EP2007421A2/fr
Priority to JP2007558260A priority patent/JP2008531722A/ja
Priority to CA002600036A priority patent/CA2600036A1/fr
Priority to NZ561144A priority patent/NZ561144A/en
Priority to AU2006220835A priority patent/AU2006220835B2/en
Publication of WO2006096497A2 publication Critical patent/WO2006096497A2/fr
Publication of WO2006096497A3 publication Critical patent/WO2006096497A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A61K39/36—Allergens from pollen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6006—Cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6025—Nucleotides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Immune responses to resolve different pathologies are important to the overall health of the host. Successful resolution of infections, cancer, or allergic reactions may depend on the type and magnitude of the immune response. Immunizations, whereby antigen is used to elicit further immune responses, may be helpful in successfully resolving the infections, cancers, and/or allergic reactions.
  • Immunostimulatory polynucleotide- immunomodulatory molecule conjugate compositions are disclosed in WO 98/16247.
  • Immunomodulatory compositions containing an immunostimulatory sequence linked to antigens are disclosed in WO 01/35991. Methods of modulating an immune response using immunostimulatory sequences are disclosed in WO 01/12223.
  • compositions comprising a structurally stable conjugate molecule, wherein the conjugate molecule comprises a conjugate partner and a polynucleotide comprising an immunostimulatory sequence (ISS) and wherein the composition further comprises a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0.
  • the conjugate partner is an antigen, such as for example, an allergen.
  • the allergen is selected from the group consisting of Crustacea allergens, insect allergens, mammalian allergens, mollusks allergens, plant allergens, and fungal allergens.
  • the allergen is the plant allergen Ragweed antigen Amb a 1.
  • a conjugate molecule is AIC, as described herein.
  • the composition comprises more than about 70% of said conjugate molecule in non-aggregate form at a temperature of between about 2 degrees C and about 8 degrees C as measured by Right Angle Light Scatter (RALS).
  • the composition comprises more than about 80%, more than about 90%, more than 95%, or more than about 97% of said conjugate molecule in non-aggregate form at a temperature of between about 2 degrees C and about 8 degrees C as measured by Right Angle Light Scatter (RALS).
  • a composition comprising a conjugate partner comprises a component capable of maintaining the pH selected from the group consisting of non-polar components and non-negatively charged components.
  • a component capable of maintaining the pH is Histidine.
  • the Histidine is present in the composition at a concentration of between about ImM and about 5OmM.
  • the Histidine is present in the composition at a concentration of between about 5mM and about 2OmM.
  • the component capable of maintaining the pH is phosphate.
  • the phosphate is present in the composition at a concentration of between about 5mM and about 5OmM and in other examples, is between about 2OmM and about 5OmM.
  • the composition has a pH in the range of about 6.0 to about 9.0.
  • the composition has a pH in the range of about 7.0 to about 8.0; and in other examples, in the range of about 7.5 to about 8.0.
  • a conjugate molecule comprises an ISS that comprises the hexamer motif AACGTT.
  • an ISS comprises the motif GACGCTCC; GACGTCCC; GACGTTCC; GACGCCCC; AGCGTTCC; AGCGTCCC; AGCGCCCC; AACGTCCC; AACGCCCC; AACGTTCC; AACGCTCC; GGCGTTCC; GGCGTCCC; GGCGCCCC; GACGCTCG; GACGTCCG; GACGCCCG; GACGTTCG; AGCGCTCG; AGCGTTCG; AGCGTCCG; AGCGCCCG; AGCGCCCG; AACGTCCG; AACGCCCG; AACGTTCG; AACGCTCG; GGCGTTCG; GGCGCTCG; GGCGCTCG; GGCGTCCG; GGCGCCCG GACGCT; GACGTC; GACGCT; GACGTC; GACGCT; GACGTC; GACGCT; GACGTC; GACGCT; GACGTC; GACGCT; GACGTC; GACGCT; GACGTC; GACGCT; GACGTC; GACGCT; GACGTC; GA
  • an ISS comprises the sequence 5'-TGACTGTGAACGTTCGAGATGA-S' (SEQ ID NO: 1); 5'-TGACCGTGAACGTTCGAGATGA-S' (SEQ ID NO:2); 5'-TCATCTCGAACGTTCCACAGTCA-S' (SEQ ID NO:3); 5'-TGACTGTGAACGTTCCAGATGA-S' (SEQ ID NO:4); 5'-TCCATAACGTTCGCCTAACGTTCGTC-S' (SEQ ID NO:5); 5'-TGACTGTGAABGTTCCAGATGA-S' (SEQ ID NO:6); 5'-TGACTGTGAABGTTCGAGATGA-S' (SEQ ID NO:7); or
  • an ISS comprises 5'-TGACTGTGAACGTTCGAGATGA-S'.
  • the conjugate partner is an antigen and yet in other examples is an allergen.
  • the allergen is selected from the group consisting of Crustacea allergens, insect allergens, mammalian allergens, mollusks allergens, plant allergens, and fungal allergens.
  • the allergen is the plant allergen Ragweed antigen Amb a 1.
  • a composition comprising a conjugate partner further comprises an amino acid selected from the group consisting of Histidine, Glycine, Isoleucine, Leucine, Proline and Alanine.
  • the amino acid is Glycine; in some examples, the Glycine is present in the composition at a concentration of between about 23OmM and about 285mM.
  • a composition further comprises a carbohydrate selected from the group consisting of Lactose, Sucrose, Mannose, Maltose, Sorbitol, and Glucose.
  • the carbohydrate is Sucrose.
  • the Sucrose is present in the composition at a concentration of between about 1% and 10%.
  • the carbohydrate is Sorbitol.
  • the Sorbitol is present in the composition at a concentration of between about 3% and about 5%.
  • compositions comprising a conjugate molecule; Histidine at a concentration in the range of about ImM to about 5OmM; and Glycine at a concentration in the range of about 5OmM to about 30OmM, wherein said composition has a pH in the range of about 6.0 to about 9.0.
  • compositions comprising a conjugate molecule further comprising Sorbitol in the range of about 1 to 10% or Sucrose at a concentration of about 20OmM to about 25OmM.
  • a composition has a pH in the range of about 7.0 to about 8.0.
  • compositions comprising a conjugate molecule, 5mM Histidine and 285 mM Glycine at a pH range of between about 7.0 and about 8.0.
  • compositions comprising a conjugate partner, 2OmM Histidine and 270 mM Glycine at a pH range of between about 7.0 and about 8.0.
  • compositions comprising 2OmM Histidine, 5OmM Glycine, and 3.8% Sorbitol at a pH range of between about 7.0 and about 8.0.
  • compositions comprising 20 mM Histidine, 50 mM Glycine, and 210 mM Sucrose at a pH range of between about 7.0 and about 8.0.
  • the present invention provides compositions in liquid form, lyophilized form, and a liquid form reconstituted from a lyophilized form.
  • the present invention also provides methods for making and using compositions described herein that comprise structurally stable conjugate molecules, such as for example, but not limited to AIC, and kits and articles of manufacture that comprise such compositions.
  • FIG. 1 shows the effect of ionic strength, time and temperature on AIC as determined by RALS as discussed in Example 5.
  • Control is 1OmM Sodium Phosphate, 141.7 mM NaCl, pH 7.2. (The bars are from left to right, t ⁇ , t7 3OC, Xl 4OC, tl4 30C, for each of the control and NaCl concentrations).
  • FIGS. 2A-2C show SEC-HPLC Chromatograms of AIC in 0.1M NaCl Base Buffer (BB) at t 7 (30 degrees C) as discussed in Example 5.
  • Fig. 2A is 215 nm;
  • Fig. 2B is 260 nm;
  • Fig. 2C is 280 nm.
  • FIG. 3 shows the effect of pH, time and temperature on AIC as measured by Extrinsic Fluorescence as discussed in Example 5.
  • Control is 1OmM Sodium Phosphate, 141.7 mM NaCl, pH 7.2. (The bars are from left to right, t ⁇ , t7-30, t7-40 5 tl4-30, for each pH value).
  • FIGS. 4A-4C Fig. 4 A shows the effect of pH on AIC: t 0 SEC-HPLC (215 nm).
  • Fig 4B shows the effect of pH, time and temperature (40°) on AIC: t 7 SEC-HPLC (215 nm).
  • Fig 4C shows the effect of pH, time and temperature (30°C) on AIC: t 14 SEC- HPLC (215 nm).
  • Control is 1OmM Sodium Phosphate, 141.7 mM NaCl, pH 7.2.
  • %MRc is % monomer recovery;
  • % Tot Rc is % total recovery; and
  • M Rec tO is monomer recovery at time 0.
  • the bars are from left to right % Non-aggregation, %MRc, and % Tot Rc for each of CtI (control), PBS control and each pH value.
  • Figures 4B-4C the bars are from left to right, % Non-aggregation, %MRc, % Tot Rc; and M Rec t0 for each of CtI (control), PBS control and each pH value).
  • FIG. 5 shows the effect of combinations of components, time and temperature on AIC as measured by RALS as shown in the Examples.
  • PBS Control is 1OmM Sodium Phosphate, 141.7 mM NaCl, pH 7.2. (The bars are from left to right, tO, XJ 3 tl4 and t28, for each composition).
  • compositions comprising a conjugate molecule, wherein the conjugate molecule comprises a conjugate partner and a polynucleotide comprising an immunostimulatory sequence (ISS).
  • ISS immunostimulatory sequence
  • the present invention is based, in part, upon the finding that the structural stability of a conjugate molecule within a composition is dependent upon temperature, salt and pH conditions.
  • the present inventors have found that a conjugate molecule comprising an antigen undergoes aggregation with time when stored liquid at 2-8 degrees C in compositions comprising sodium phosphate and sodium chloride. It was also found that low pH caused a conjugate molecule comprising an antigen to aggregate reversibly under conditions of low ionic strength.
  • compositions developed to minimize or reduce aggregation of conjugate molecules and methods of making and using such compositions. Described herein are compositions comprising a conjugate molecule that is structurally stable at a temperature of between about 2 degrees C to about 8 degrees C.
  • the conjugate molecule comprises an antigen, such as for example, an allergen, hi some examples, the allergen is purified short ragweed antigen (Amb a 1).
  • an antigen such as for example, an allergen
  • the allergen is purified short ragweed antigen (Amb a 1).
  • compositions comprising a conjugate molecule that comprises Amb a 1 that is structurally stable at about 2 to about 8% C.
  • compositions comprising a conjugate molecule and a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0.
  • the composition comprises more than 70%, more than 80%, more than 90%, more than 95%, more than 97%, more than 98% or more than 99% of said conjugate molecule in non-aggregate form at a temperature of between about 2 degrees C and about 8 degrees C.
  • the composition comprises more than 70%, more than 80%, more then 90%, more then 95% or more than 97% of said conjugate molecule in non-aggregate form, for a period of up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 6 weeks, up to about 8 weeks, up to about 10 weeks, up to about 12 weeks, up to about 14 weeks, up to about 16 weeks, up to about 18 weeks, up to about 20 weeks, up to about 22 weeks, up to about 24 weeks, up to about 1 year or up to about 2 years.
  • aggregation is measured by Right Angle Light Scatter (RALS).
  • aggregation of a conjugate molecule in a composition is measured by intrinsic fluorescence (IF), extrinsic fluorescence (EF) and/or SEC-HPLC which may or may not be in combination with RALS.
  • the conjugate molecule comprises an antigen.
  • antigens are known in the art and include but are not limited to peptides, lipids, polysaccharides, gangliosides, and glycoproteins.
  • the antigen is an allergen.
  • allergens examples include, but are not limited to, Crustacea allergens, insect allergens, mammalian allergens, mollusks allergens, plant allergens, and fungal allergens.
  • the allergen is a plant allergen, such as for example, Ragweed antigen.
  • the allergen is Amb a 1.
  • compositions comprising a conjugate molecule that comprises Amb a 1 that further comprises a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0, wherein the composition comprises more than about 70%, more than about 80%, more than about 90%, more than about 95% or more than about 97% of said conjugate molecule in non-aggregate form at a temperature of between about 2 degrees C and about 8 degrees C.
  • a composition comprising a conjugate molecule may further comprise one or more of 1) an amino acid, 2) a carbohydrate, 3) a surfactant, or 4) other suitable component as long as the composition comprises more than 70%, more than about 80%, more than about 90%, more than about 95% or more than about 97% of said conjugate molecule comprising Amb a 1 in non-aggregate form.
  • the composition is in liquid form, m other examples, the composition is lyophilized and in yet other examples, the composition is in liquid form that has been reconstituted from a lyophilized form.
  • compositions comprising a conjugate molecule wherein less than about 30%, less than about 20%, less than about 10%, less than about 5% or less than about 3% of the conjugate molecule present in the composition is in aggregate form at a temperature between about 2 degrees C and about 8 degrees C.
  • less than about 30%, less than about 20%, less than about 10%, less than about 5% or less than about 3% of the conjugate molecule present in the composition is in aggregate form at a temperature between about 2 degrees C and about 8 degrees C for a period of up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 4 weeks, up to about 6 weeks, up to about 8 weeks, up to about 10 weeks, up to about 12 weeks, up to about 14 weeks, up to about 16 weeks, up to about 18 weeks, up to about 20 weeks, up to about 22 weeks, up to about 24 weeks, up to about 1 year or up to about 2 years.
  • aggregation of a conjugate molecule in a composition is measured by right angle light scatter (RALS).
  • the conjugate molecule comprises an antigen.
  • the antigens are peptides, lipids (e.g., sterols excluding cholesterol, fatty acids, and phospholipids), polysaccharides, gangliosides and glycoproteins.
  • the antigens include, but are not limited to antigens from an infectious agent, including protozoan, bacterial, fungal (including unicellular and multicellular), and viral infectious agents.
  • antigens from parasitic organisms include schistosome egg antigens (e.g., Sm-p40) from Schistosome species (e.g., S. mansoni) and antigens from Toxoplasma species (e.g., T.
  • the antigen is an allergen.
  • allergens are known in the art and described herein and include, but are not limited to, Crustacea allergens, insect allergens, mammalian allergens, mollusks allergens, plant allergens, and fungal allergens.
  • the allergen is a plant allergen, such as for example, Ragweed antigen.
  • the allergen is Amb a 1.
  • compositions comprising a conjugate molecule comprising Amb al wherein less than about 30%, less than about 20%, less than about 10%, less than about 5% or less than about 3% of the conjugate molecule present in the composition is in aggregate form at a temperature between about 2 degrees C and about 8 degrees C.
  • compositions comprising a structurally stable conjugate molecule as described herein.
  • methods for modulating the immune response of a mammalian host that comprise administration of a composition comprising a structurally stable conjugate molecule as described herein to a mammalian host; pharmaceutical compositions comprising a structurally stable conjugate molecule; kits comprising compositions comprising a structurally stable conjugate molecule; and articles of manufacture comprising a composition comprising a structurally stable conjugate molecule.
  • an article of manufacture comprises a composition comprising a conjugate molecule wherein the composition comprises greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95% or greater than about 97% of the conjugate molecule in non-aggregate form at a temperature of between about 2 degrees C and about 8 degrees C. In some examples, aggregation is measured by RALS.
  • an article of manufacture comprises a liquid composition comprising a conjugate molecule and in other examples an article of manufacture comprises a lyophilized composition comprising a conjugate molecule.
  • an article of manufacture comprises a reconstituted liquid composition (reconstituted from a lyophilized composition) comprising a structurally stable conjugate molecule.
  • an ISS includes one or more ISS.
  • the term "immunostimulatory sequence” or "ISS” as used herein refers to polynucleotide sequences that effect a measurable immune response as measured in vitro, in vivo and/or ex vivo. Examples of measurable immune responses include, but are not limited to, antigen-specific antibody production, secretion of cytokines, activation or expansion of lymphocyte populations such as NK cells, CD4 + T lymphocytes, CD8 + T lymphocytes, B lymphocytes, and the like.
  • the ISS sequences preferentially activate a ThI -type response.
  • a polynucleotide for use in the invention contains at least one ISS.
  • ISS is also a shorthand term for an ISS-containing polynucleotide.
  • a "conjugate molecule” as used herein refers to a molecule or complex that comprises an ISS (that is, an ISS-containing polynucleotide) and a conjugate partner. In some examples, the ISS and conjugate partner are linked directly or indirectly. Such conjugate linkages include covalent and/or non-covalent linkages. Conjugate partners include but are not limited to antigens.
  • a "population of conjugate molecules” is a group of ISS-conjugate partners (i.e., ISS directly or indirectly linked, or attached, to conjugate partner).
  • populations do not necessarily have, and may or may not have, a constant number of ISS attached to each conjugate partner.
  • a given population will have a distribution of molecular weights (based on varying extent of conjugation within a given population) and thus an average number of ISS conjugated to the conjugate partner.
  • any of the populations of conjugate molecules described herein may contain molecules of free conjugate partners (i.e., conjugate partner not linked to ISS) and/or free ISS (i.e., ISS not linked to conjugate partner), due to, for example, incomplete conjugation and/or purification.
  • the populations described herein contain conjugate molecules, but need not exclusively contain conjugate molecules.
  • an "AIC" conjugate molecule refers to the Ragweed allergen, Amb a 1, conjugated to an ISS.
  • Structural stability of a conjugate molecule within a composition refers to a composition in which the conjugate molecule essentially retains it physical stability and integrity. Physical stability of a conjugate molecule within a composition is measured by the amount, that is, percentage (%), of aggregation of the conjugate molecule in the composition. Generally, increased % of aggregation of conjugate molecules within a composition is correlated with decreased structurally stability of conjugate molecules within a composition.
  • Structural stability of a conjugate molecule within a compositions does not require that there is 0% aggregation of conjugate molecules within the composition, or 0% aggregation of free, that is, non-conjugated, conjugate partners, if present in the composition, or 0% aggregation of free, that is, non-conjugated ISS, if present in the composition.
  • a composition comprising a conjugate molecule that is structurally stable refers to a composition wherein greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95% or greater than about 97 % of the conjugate molecule is present in non-aggregate form in the composition.
  • non-aggregate form with respect to a conjugate molecule includes but is not limited to the conjugate molecule in monomer form.
  • increased % of monomers of conjugate molecules within a composition is correlated with increased structurally stability of the conjugate molecules within a composition.
  • a composition comprising a conjugate molecule that is structurally stable refers to, that is includes, a composition wherein at least about 70%, at least about 80%, at least about 90%, at least 95% and at least about 97% of the conjugate molecule is present in the composition in monomer form at a temperature between about 2 degrees C and about 8 degrees C.
  • a composition comprising a conjugate molecule that is structurally stable refers to, that is includes, a composition wherein less than about 30%, less than about 20%, less than about 10%, less than about 5% or less than about 3 % of the conjugate molecule is present as an aggregate in the composition.
  • Various methods for measuring structural stability are available in the art and are disclosed herein and include but are not limited to Right Angle Light Scatter (RALS), either alone or in combination with other methods known in the art and described herein, including but not limited to IF, EF and SEC-HPLC.
  • % aggregation of a conjugate molecule within a composition is measured by RALS.
  • a composition comprising a conjugate molecule that is "destabilized” is one that has greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95% of the conjugate molecule in the composition present in aggregate form.
  • An "average" of a given parameter (such as number of ISS-containing polynucleotides or mass) in a given population means the total of that parameter for the entire population divided by the number of members of the population.
  • the average number of ISS-containing polynucleotides attached to conjugate partner refers to the average number of ISS-containing polynucleotides per conjugate partner in a population of conjugate partner molecules (i.e., total number of ISS-containing polynucleotides divided by total number of conjugate partners). As described below, this number is usually derived from weight determinations of polynucleotide to conjugate partner, as measured, for example, by spectroscopy.
  • a “median" number or weight for a given population refers to a number or weight at which half the population is above, and half the population is below.
  • a median number of ISS-containing polynucleotides per conjugate partner means that half the conjugate partners in the population have a lower number of ISS-containing polynucleotides per conjugate partner, and half have a higher number.
  • oligonucleotide include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), modified oligonucleotides and oligonucleosides or combinations thereof.
  • the oligonucleotide can be linearly or circularly configured, or the oligonucleotide can contain both linear and circular segments.
  • Oligonucleotides are polymers of nucleosides joined, generally, through phosphoester linkages.
  • a nucleoside consists of a purine (adenine or guanine or derivative thereof) or pyrimidine (thymine, cytosine or uracil, or derivative thereof) base bonded to a sugar.
  • the four nucleoside units (or bases) in DNA are called deoxyadenosine, deoxyguanosine, deoxythymidine, and deoxycytidine.
  • a nucleotide is a phosphate ester of a nucleoside.
  • immunomodulatory or “modulating an immune response” as used herein includes immunostimulatory as well as immunosuppressive effects. Immunostimulatory effects include, but are not limited to, those that directly or indirectly enhance cellular or humoral immune responses.
  • immunostimulatory effects include, but are not limited to, increased antigen-specific antibody production; activation or proliferation of a lymphocyte population such as NK cells, CD4 T lymphocytes, CD8 T lymphocytes, macrophages and the like; increased synthesis of immunostimulatory cytokines including, but not limited to, IL-I, IL-2, IL-4, IL-5, IL-6, IL-10, IL- 12, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , TNF- ⁇ and the like.
  • Immunosuppressive effects include those that directly or indirectly decrease cellular or humoral immune responses.
  • immunosuppressive effects include, but are not limited to, a reduction in antigen-specific antibody production such as reduced IgE production; activation of lymphocyte or other cell populations that have immunosuppressive activities such as those that result in immune tolerance; and increased synthesis of cytokines that have suppressive effects toward certain cellular functions.
  • IFN- ⁇ which appears to block IL-4 induced class switch to IgE and IgGl, thereby reducing the levels of these antibody subclasses.
  • Extent of conjugation means the average degree of conjugation in a given population. As described herein, extent of conjugation may be characterized by any of a number of structural and/or functional parameters, either alone or in any combination.
  • antigen means a substance that is recognized and bound specifically by an antibody or by a T cell antigen receptor.
  • Antigens can include peptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids and phospholipids; portions thereof and combinations thereof.
  • the antigens can be those found in nature or can be synthetic.
  • Antigens suitable for administration with ISS include any molecule capable of eliciting a B cell or T cell antigen-specific response. Preferably, antigens elicit an antibody response specific for the antigen.
  • Haptens are included within the scope of "antigen.”
  • a hapten is a low molecular weight compound that is not immunogenic by itself but is rendered immunogenic when conjugated with an immunogenic molecule containing antigenic determinants. Small molecules may need to be haptenized in order to be rendered antigenic.
  • antigens of the present invention include peptides, lipids (e.g. sterols, fatty acids, and phospholipids), polysaccharides such as those used in Hemophilus influenza vaccines, gangliosides and glycoproteins.
  • Adjuvant refers to a substance which, when added to an immunogenic agent such as antigen, nonspecifically enhances or potentiates an immune response to the agent in the recipient host upon exposure to the mixture.
  • peptide are polypeptides that are of sufficient length and composition to effect a biological response, e.g. antibody production or cytokine activity whether or not the peptide is a hapten. Typically, the peptides are of at least six amino acid residues in length.
  • the term “peptide” further includes modified amino acids (whether or not naturally or non-naturally occurring), such modifications including, but not limited to, phosphorylation, glycosylation, pegylation, lipidization and methylation.
  • Antigenic peptides can include purified native peptides, synthetic peptides, recombinant proteins, crude protein extracts, attenuated or inactivated viruses, cells, microorganisms, or fragments of such peptides.
  • An "antigenic peptide” or “antigen polypeptide” accordingly means all or a portion of a polypeptide which exhibits one or more antigenic properties.
  • an "Amb a 1 antigenic polypeptide” or “Amb a 1 polypeptide antigen” is an amino acid sequence from Amb a 1, whether the entire sequence, a portion of the sequence, and/or a modification of the sequence, which exhibits an antigenic property ⁇ i.e., binds specifically to an antibody or a T cell receptor).
  • a "delivery molecule” or “delivery vehicle” is a chemical moiety which facilitates, permits, and/or enhances delivery of an ISS and/or antigen to a particular site and/or with respect to particular timing.
  • a delivery vehicle may or may not additionally stimulate an immune response.
  • An "allergic response to antigen” means an immune response generally characterized by the generation of eosinophils and/or antigen-specific IgE and their resultant effects. As is well-known in the art, IgE binds to IgE receptors on mast cells and basophils.
  • the antigen Upon later exposure to the antigen recognized by the IgE, the antigen crosslinks the IgE on the mast cells and basophils causing degranulation of these cells, including, but not limited, to histamine release.
  • the terms "allergic response to antigen”, “allergy”, and “allergic condition” are equally appropriate for application of some of the methods of the invention. Further, it is understood and intended that the methods of the invention include those that are equally appropriate for prevention of an allergic response as well as treating a pre-existing allergic condition.
  • allergen means an antigen or antigenic portion of a molecule, usually a protein, which elicits an allergic response upon exposure to a subject.
  • the subject is allergic to the allergen as indicated, for instance, by the wheal and flare test or any method known in the art.
  • a molecule is said to be an allergen even if only a small subset of subjects exhibit an allergic ⁇ e.g., IgE) immune response upon exposure to the molecule.
  • IgE allergic ⁇ e.g., IgE
  • isolated allergens are known in the art. These include, but are not limited to, those provided herein.
  • desensitization refers to the process of the administration of increasing doses of an allergen to which the subject has demonstrated sensitivity.
  • allergen doses used for desensitization are known in the art, see, for example, Fornadley (1998) Otolaryngol Clin. North Am. 31 : 111 -127.
  • Antigen-specific immunotherapy refers to any form of immunotherapy which involves antigen and generates an antigen-specific modulation of the immune response.
  • antigen-specific immunotherapy includes, but is not limited to, desensitization therapy.
  • mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets.
  • an “effective amount” or a “sufficient amount” of a substance is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an "effective amount” depends upon the context in which it is being applied.
  • an effective amount of a composition comprising an ISS-conjugate partner that is, an ISS and conjugate partner
  • an effective amount is an amount sufficient to achieve such a modulation as compared to the immune response obtained when the antigen is administered alone.
  • An effective amount can be administered in one or more administrations.
  • co-administration refers to the administration of at least two different substances sufficiently close in time to modulate an immune response.
  • co-administration refers to simultaneous administration of at least two different substances.
  • Stimulation of an immune response means an increase in the response, which can arise from eliciting and/or enhancement of a response.
  • An "allergy-related disorder” means a disorder resulting from the effects of an antigen-specific IgE immune response. Such effects can include, but are not limited to, hypotension and shock.
  • Anaphylaxis is an example of an allergy-related disorder during which histamine released into the circulation causes vasodilation as well as increased permeability of the capillaries with resultant marked loss of plasma from the circulation. Anaphylaxis can occur systemically, with the associated effects experienced over the entire body, and it can occur locally, with the reaction limited to a specific target tissue or organ.
  • IgE associated disorder is a physiological condition which is characterized, in part, by elevated IgE levels, which may or may not be persistent.
  • IgE associated disorders include, but are not limited to, allergy and allergic reactions, allergy- related disorders (described below), asthma, rhinitis, conjunctivitis, urticaria, shock, hymenoptera sting allergies, and drug allergies, and parasite infections. The term also includes related manifestations of these disorders. Generally, IgE in such disorders is antigen-specific.
  • treatment is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • “Palliating" a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.
  • palliation may occur upon modulation of the immune response against an allergen(s). Further, palliation does not necessarily occur by administration of one dose, but often occurs upon administration of a series of doses. Thus, an amount sufficient to palliate a response or disorder may be administered in one or more administrations.
  • an “antibody titer”, or “amount of antibody”, which is “elicited” by an ISS- conjugate partner or ISS-antigen refers to the amount of a given antibody measured at a time point after administration of conjugate or antigen.
  • a “ThI -associated antibody” is an antibody whose production and/or increase is associated with a ThI immune response.
  • IgG2a is a ThI -associated antibody in mouse.
  • measurement of a ThI -associated antibody can be a measurement of one or more such antibodies.
  • measurement of a ThI -associated antibody could entail measurement of IgGl and/or IgG3.
  • Th2-associated antibody is an antibody whose production and/or increase is associated with a Th2 immune response.
  • IgGl is a Th2-associated antibody in mouse.
  • measurement of a Th2-associated antibody can be measurement of one or more such antibodies.
  • measurement of a Th2-associated antibody could entail measurement of IgG2 and/or IgG4.
  • a function or activity such as cytokine production, antibody production, or histamine release, is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
  • a conjugate molecule population which suppresses histamine release reduces histamine release as compared to, for example, histamine release induced by antigen alone.
  • a conjugate molecule population which suppresses antibody production reduces extent and/or levels of antibody as compared to, for example, extent and/or levels of antibody produced by antigen alone.
  • compositions comprising conjugate molecules
  • the present invention relates, in part, to compositions comprising a conjugate molecule, wherein the conjugate molecule comprises a conjugate partner and a polynucleotide comprising an immunostimulatory sequence (ISS) and, in part, to methods of making and using such compositions.
  • the present invention also relates, in part, to compositions comprising structurally stable conjugate molecules and methods of making and using such compositions.
  • ISS and conjugate partners encompassed within the present invention are described herein.
  • conjugate molecules encompassed within the present invention may have differing and distinct biological properties, based on the conjugate partner, the type and number of ISS present in the conjugate molecule and the average extent of conjugation between the ISS and conjugate partner.
  • a conjugate partner is a protein, such as an antigen or an allergen.
  • the conjugate partner comprises the allergen, Amb a 1.
  • the ISS can be a polynucleotide of any length greater than 6 bases or base pairs and in some examples comprises the sequence 5'-cytosine (C), guanine-3' (G), and in other examples comprises the sequence 5 '-purine, purine, C, G, pyrimidine, pyrimidine-3' (such as 5'-AACGTT-3'), and in other examples is greater than 15 bases or base pairs, and in other examples is greater than 20 bases or base pairs in length.
  • an ISS comprises the sequence 5 '-purine, purine, C, G, pyrimidine, pyrimidine, C, G-3'; or the sequence 5 '-purine, purine, C, G, pyrimidine, pyrimidine, C, C-3'; or the sequence 5'-T, C, G-3'.
  • the ISS comprises 5 '-TGACTGTGAACGTTCGAGATGA-S'.
  • the conjugate molecule comprises Amb a 1 conjugated to an ISS comprising 5'-TGACTGTGAACGTTCGAGATGA-S'.
  • ISS immunostimulatory sequence
  • the inventors discovered that the structural stability of a conjugate molecule within a composition is dependent upon temperature, salt and pH conditions. It was found that a conjugate molecule comprising an allergen was destabilized in a liquid composition at temperatures above 0 degrees C or higher by the presence of sodium phosphate, sodium chloride, negatively charged components and/or polar components in a composition. It was found that low pH caused a conjugate molecule comprising an allergen to aggregate reversibly under conditions of low ionic strength.
  • compositions comprising non- negatively charged components or components having a neutral charge or non-polar components are desired to maintain the structural stability of the conjugate molecule present in the composition. It has been found that the addition of components having a negative charge, such as sodium chloride, will cause the conjugate molecules comprising an ISS to aggregate, thereby becoming destabilized. It has been found that the presence of nucleophiles, such as azides, or even low sodium chloride in a composition comprising a conjugate molecule will destabilize the conjugate molecule.
  • compositions The structural stability of a conjugate molecule comprising an allergen within various compositions was characterized as described herein. Experiments were designed to elucidate possible structural changes in conjugate molecules within compositions as a function of pH, temperature, time and composition conditions, such as ionic strength, and presence of surfactants.
  • an illustrative example of a conjugate molecule comprising a purified short ragweed antigen, AMB a 1, covalently linked to an ISS (referred to herein as Amb a 1-ISS Oligonucleotide Conjugate or "AIC") within a concentration range of about 0.1 fig to 200 ⁇ .g was dialyzed into compositions containing varying ionic strengths and varying pH conditions and then analyzed by using intrinsic fluorescence (IF); extrinsic fluorescence (EF); and right angle light scatter (RALS) while subjecting the conjugate molecule to temperature variations.
  • IF, EF, and RALS are described herein in the Examples.
  • the conjugate molecule was subjected to shear stress and analyzed by RALS and size exclusion chromatograph HPLC (SEC-HPLC).
  • SEC-HPLC size exclusion chromatograph HPLC
  • the same methods, that is, IF, EF, RALS, and SEC-HPLC, were used in parallel to examine freeze- thaw sensitivity of a composition comprising AIC in the presence of sodium phosphate and sodium chloride (referred to herein in the Examples as PBS).
  • Table 10 shows the results of analysis of various components for maintaining pH conditions.
  • components for maintaining pH conditions of a composition comprising a conjugate molecule include Histidine at pH 7.5, Histidine at pH 8.0, Phosphate at pH 7.5, Phosphate at pH 8.0, Phosphate at pH 7.0, Histidine at pH 7.0 or Histidine at pH 6.5.
  • compositions were prepared that comprised Histidine.
  • Table 11 shows the results of analysis of various amino acids.
  • amino acids for use in a composition comprising a conjugate molecule include Histidine, Glycine, Isoleucine, Leucine, Proline, or Alanine.
  • compositions were prepared that comprised Glycine.
  • Table 12 shows the results of analysis of various carbohydrates.
  • a composition comprising a conjugate molecule includes a carbohydrate, such as Lactose, Sucrose, Mannose or Maltose. Example 6 describes results of combination studies.
  • compositions comprising a structurally stable conjugate molecule at a temperature of between about 2 degrees C and about 8 degrees C.
  • the composition is in liquid form and in other examples is in a lyophilized form.
  • compositions comprising a structurally stable conjugate molecule and a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0.
  • the component is capable of maintaining the pH in the range of about 7.0 to about 8.0, and in other examples, in the range of about 7.5 to about 7.8, and in other examples, at a pH of about 7.5.
  • the component capable of maintaining the pH in the range of about 6.0 to about 9.0 has a neutral charge or basic charge.
  • the component capable of maintaining the pH in the range of about 6.0 to about 9.0 is non-polar.
  • a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0 is selected from the group consisting of Histidine or Phosphate.
  • the component capable of maintaining the pH is present in the composition in an amount sufficient to retain more than 70%, more than 80%, more then 90%, more then 95% or more than 97% of said conjugate molecule in non-aggregate form at a temperature of between about 2 degrees C and about 8 degrees C. Aggregation of conjugate molecules can be measured by methods disclosed herein, such as by Right Angle Light Scatter (RALS), and by methods known in the art.
  • RALS Right Angle Light Scatter
  • a composition comprising a conjugate molecule may further comprise one or more of 1) an amino acid, 2) a carbohydrate, 3) a surfactant, and 4) other suitable pharmaceutically acceptable carriers as long as the composition comprises more than 70%, more than 80%, more then 90%, more then 95% or more than 97% of said conjugate molecule in non-aggregate or monomer form.
  • the amino acid is selected from the group consisting of Histidine, Glycine, Isoleucine, Leucine, Proline and Alanine. In some examples, the amino acid is Histidine or Glycine.
  • the carbohydrate is selected from the group consisting of Lactose, Sucrose, Mannose, Maltose, Sorbitol and Glucose. In some examples, the carbohydrate is Sorbitol or Sucrose.
  • compositions comprising a structurally stable conjugate molecule and a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0.
  • compositions comprising a structurally stable conjugate molecule, a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0, and an amino acid.
  • compositions comprising a structurally stable conjugate molecule, a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0, and a carbohydrate.
  • compositions comprising a structurally stable conjugate molecule, a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0, an amino acid and a carbohydrate.
  • a composition comprising a structurally stable conjugate molecule further comprises a surfactant.
  • the composition is in liquid form and in other examples is in lyophilized form.
  • the present invention encompasses compositions comprising conjugate molecules reconstituted in liquid form from a lyophilized form.
  • compositions in liquid form comprising AIC as described herein at concentrations including about 30 ug/ml and about 60 ug/ml, and comprising an average of about 4.0 moles of ISS per mole of Amb 1 a (average molecular weight of about 65kDA) identified compositions that maintain structural stability of the AIC at a temperature of between about 2 degrees C and 8 degrees C. In some examples, the structural stability was maintained for at least 6 months.
  • compositions comprising AIC include, but are not limited to compositions comprising Histidine in the range of about 1 rnM to about 50 mM; compositions comprising Histidine in the range of about 1 mM to about 50 mM and Glycine in the range of about 50 mM to about 300 mM; compositions comprising Histidine in the range of about 1 mM to about 50 mM and Sorbitol in the range of about 1% to about 5% of the composition; compositions comprising Histidine in the range of about 1 mM to about 50 mM, Glycine in the range of about 50 mM to about 300 mM, and Sorbitol in the range of about 1% to about 5% of the composition, all of which compositions have a pH in the range of about 7.0 to about 8.0.
  • compositions comprising AIC (which may be low (L), medium(M) or high(H), as described herein) wherein the AIC within the composition is structurally stable at a temperature of between about 2 degrees C. and 8 degrees C which include, but are not limited to:
  • compositions of AIC that are predicted to be structurally stable are shown below.
  • IF, EF and RALS in conjunction with analytical HPLC methods can be designed to profile the behavior of any conjugate molecule within a composition as a function of pH, temperature and time as well as solution conditions (i.e. ionic strength, surfactants, etc.) to determine conditions for structural stability of a conjugate molecule within a composition.
  • analytical HPLC methods such as, Size Exclusion (SEC-HPLC) and SDS-PAGE, for example, can be designed to profile the behavior of any conjugate molecule within a composition as a function of pH, temperature and time as well as solution conditions (i.e. ionic strength, surfactants, etc.) to determine conditions for structural stability of a conjugate molecule within a composition.
  • Conjugate partn ers Conjugate partners encompassed within the present invention include but are not limited to antigens, such as peptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids and phospholipids; portions thereof and combinations thereof.
  • the antigen can be from an infectious agent, including protozoan, bacterial, fungal (including unicellular and multicellular), and viral infectious agents.
  • antigens from parasitic organisms include schistosome egg antigens ⁇ e.g., Sm-p40) from Schistosome species (e.g., S. mansoni) and antigens from Toxoplasma species ⁇ e.g., T. gondii).
  • Antigens may be isolated from their source using purification techniques known in the art or, more conveniently, may be produced using recombinant methods.
  • Antigenic peptides can include purified native peptides, synthetic peptides, recombinant proteins, crude protein extracts, attenuated or inactivated viruses, cells, micro-organisms, or fragments of such peptides.
  • Immunomodulatory peptides can be native or synthesized chemically or enzymatically. Any method of chemical synthesis known in the art is suitable.
  • Solution phase peptide synthesis can be used to construct peptides of moderate size or, for the chemical construction of peptides, solid phase synthesis can be employed.
  • Proteolytic enzymes can also be utilized to couple amino acids to produce peptides.
  • the peptide can be obtained by using the biochemical machinery of a cell, or by isolation from a biological source. Recombinant DNA techniques can be employed for the production of peptides.
  • Peptides can also be isolated using standard techniques such as affinity chromatography.
  • the antigens are peptides, lipids ⁇ e.g., sterols excluding cholesterol, fatty acids, and phospholipids), polysaccharides, gangliosides and glycoproteins. These can be obtained through several methods known in the art, including isolation and synthesis using chemical and enzymatic methods. In certain cases, such as for many sterols, fatty acids and phospholipids, the antigenic portions of the molecules are commercially available.
  • Antigens derived from infectious agents may be obtained using methods known in the art, for example, from native viral or bacterial extracts, from cells infected with the infectious agent, from purified polypeptides, from recombinantly produced polypeptides and/or as synthetic peptides.
  • the antigen is an allergen.
  • examples of recombinant allergens are provided in Table 1 and include but are not limited to Crustacea allergens, insect allergens, mammalian allergens, mollusks allergens, plant allergens, and fungal allergens.
  • the allergen is a plant allergen.
  • the allergen is the ragweed antigen Amb a 1.
  • Preparation of many allergens is well-known in the art, including, but not limited to, preparation of ragweed pollen allergen Antigen Antigen E (Amb al) (Rafhar et al. (1991) J. Biol. Chem.
  • allergens from trees are known, including allergens from birch, juniper and Japanese cedar. Preparation of protein antigens from grass pollen for in vivo administration has been reported. Malley (1989) J. Reprod. Immunol. 16:173-186.
  • the allergen is a food allergen such as peanut allergen, for example Ara h I
  • the allergen is a grass allergen such as a rye allergen, for example LoI p I.
  • Table 1 shows a list of allergens encompassed within the present invention.
  • the antigen is from an infectious agent, including protozoan, bacterial, fungal (including unicellular and multicellular), and viral infectious agents.
  • infectious agent including protozoan, bacterial, fungal (including unicellular and multicellular), and viral infectious agents.
  • suitable viral antigens are described herein and are known in the art.
  • Bacteria include Hemophilus influenza, Mycobacterium tuberculosis and Bordetella pertussis.
  • Protozoan infectious agents include malarial plasmodia, Leishmania species, Trypanosoma species and Schistosoma species.
  • Fungi include Candida albicans.
  • the antigen is a viral antigen.
  • Viral polypeptide antigens include, but are not limited to, core proteins such as HIV gag proteins (including, but not limited to, membrane anchoring (MA) protein, core capsid (CA) protein and nucleocapsid (NC) protein), HIV polymerase, influenza virus matrix (M) protein and influenza virus nucleocapsid (NP) protein.
  • core proteins such as HIV gag proteins (including, but not limited to, membrane anchoring (MA) protein, core capsid (CA) protein and nucleocapsid (NC) protein), HIV polymerase, influenza virus matrix (M) protein and influenza virus nucleocapsid (NP) protein.
  • References discussing influenza vaccination include Scherle and Gerhard (1988) Proc. Natl. Acad. ScL USA 85:4446-4450; Scherle and Gerhard (1986) J. Exp. Med. 164:1114-1128; Granoff et al. (1993) Vaccine 11:S46-51; Kodihalli et al. (1997) J
  • antigen polypeptides are group- or sub-group specific antigens, which are known for a number of infectious agents, including, but not limited to, adenovirus, herpes simplex virus, papilloma virus, respiratory syncytial virus and poxviruses.
  • immunomodulatory peptides can include tumor cells (live or irradiated), tumor cell extracts, or protein subunits of tumor antigens such as Her-2/neu, Marti, carcinoembryonic antigen (CEA), gangliosides, human milk fat globule (HMFG), mucin (MUCl), MAGE antigens, BAGE antigens, GAGE antigens, gplOO, prostate specific antigen (PSA), and tyrosinase.
  • Vaccines for immuno-based contraception can be formed by including sperm proteins administered with ISS. Lea et al. (1996) Biochim. Biophys. Acta 1307:263. Attenuated and inactivated viruses are suitable for use herein as the antigen.
  • polio virus is available as IPOL® (Pasteur Merieux Connaught) and ORMUNE® (Lederle Laboratories), hepatitis A virus as VAQT A® (Merck), measles virus as ATTENUV AX® (Merck), mumps virus as MUMPS VAX® (Merck) and rubella virus as MERUV AX® II (Merck).
  • Attenuated and inactivated viruses such as HIV-I, HIV-2, herpes simplex virus, hepatitis B virus, rotavirus, human and non-human papillomavirus and slow brain viruses can provide peptide antigens.
  • the antigen comprises a viral vector, such as vaccinia, adenovirus, and canary pox.
  • Antigens may be isolated from their source using purification techniques known in the art or, more conveniently, may be produced using recombinant methods.
  • Antigenic peptides can include purified native peptides, synthetic peptides, recombinant proteins, crude protein extracts, attenuated or inactivated viruses, cells, microorganisms, or fragments of such peptides.
  • Immunomodulatory peptides can be native or synthesized chemically or enzymatically. Any method of chemical synthesis known in the art is suitable. Solution phase peptide synthesis can be used to construct peptides of moderate size or, for the chemical construction of peptides, solid phase synthesis can be employed. Atherton et al. (1981) Hoppe Seylers Z.
  • Proteolytic enzymes can also be utilized to couple amino acids to produce peptides.
  • the peptide can be obtained by using the biochemical machinery of a cell, or by isolation from a biological source. Recombinant DNA techniques can be employed for the production of peptides. Hames et al. (1987) Transcription and Translation: A Practical Approach, IRL Press.
  • Peptides can also be isolated using standard techniques such as affinity chromatography.
  • the antigens are peptides, lipids (e.g. , sterols, fatty acids, and phospholipids), polysaccharides such as those used in H. influenza vaccines, gangliosides and glycoproteins. These can be obtained through several methods known in the art, including isolation and synthesis using chemical and enzymatic methods. In certain cases, such as for many sterols, fatty acids and phospholipids, the antigenic portions of the molecules are commercially available. Examples of viral antigens useful in the subject compositions and methods using the compositions include, but are not limited to, HIV antigens.
  • antigens include, but are not limited to, those antigens derived from HIV envelope glycoproteins including, but not limited to, gpl 60, gpl20 and gp41.
  • HIV envelope glycoproteins including, but not limited to, gpl 60, gpl20 and gp41.
  • Numerous sequences for HIV genes and antigens are known.
  • the Los Alamos National Laboratory HIV Sequence Database collects, curates and annotates HIV nucleotide and amino acid sequences. This database is accessible via the internet, at http://hiv-web.lanl.gov/, and in a yearly publication, see Human Retroviruses and AIDS Compendium (for example, 1998 edition).
  • Antigens derived from infectious agents may be obtained using methods known in the art, for example, from native viral or bacterial extracts, from cells infected with the infectious agent, from purified polypeptides, from recombinantly produced polypeptides and/or as synthetic peptides.
  • ISS native viral or bacterial extracts
  • purified polypeptides from cells infected with the infectious agent
  • purified polypeptides from recombinantly produced polypeptides and/or as synthetic peptides.
  • the conjugate molecule contains at least one ISS, and can contain multiple ISSs.
  • the ISSs can be adjacent within the polynucleotide, or they can be separated by additional nucleotide bases within the polynucleotide.
  • ISS have been described in the art and may be readily identified using standard assays which indicate various aspects of the immune response, such as cytokine secretion, antibody production, NK cell activation and T cell proliferation. See, e.g., WO 97/28259; WO 98/16247; WO 99/11275; Krieg et al. (1995) Nature 374:546-549; Yamamoto et al. (1992a); Ballas et al.
  • the ISS can be of any length greater than 6 bases or base pairs and generally comprises the sequence 5'-cytosine, guanine-3', more particularly comprises the sequence 5'-purine, purine, C, G, pyrimidine, pyrimidine-3' (such as 5'-AACGTT-3'), preferably, in some examples, greater than 15 bases or base pairs, more preferably, in some examples, greater than 20 bases or base pairs in length.
  • An ISS may also comprise the sequence 5'- purine, purine, C, G, pyrimidine, pyrimidine, C 5 G-3 ⁇
  • An ISS may also comprise the sequence 5 '-purine, purine, C, G, pyrimidine, pyrimidine, C, C-3'.
  • an ISS may also comprise the sequence 5'-T, C, G-3'.
  • the ISS comprises any of the following sequences:
  • GACGCTCC GACGTCCC; GACGTTCC; GACGCCCC; AGCGTTCC; AGCGTCCC; AGCGCCCC; AACGTCCC; AACGCCCC; AACGTTCC; AACGCTCC; GGCGTTCC; GGCGTCCC; GGCGCCCC; GACGCTCG; GACGTCCG; GACGCCCG; GACGTTCG; AGCGCTCG; AGCGTTCG; AGCGTCCG; AGCGCCCG; AGCGTCCG; AGCGCCCG; AACGTCCG; AACGCCCG; AACGTTCG; AACGCTCG; GGCGTTCG; GGCGCTCG; GGCGTCCG; GGCGCCCG.
  • the ISS comprises anyofthe following sequences: GACGCT; GACGTC; GACGTT; GACGCC; GACGCU; GACGUC; GACGUU; GACGUT; GACGTU; AGCGTT; AGCGCT; AGCGTC; AGCGCC; AGCGUU; AGCGCU; AGCGUC; AGCGUT; AGCGTU; AACGTC; AACGCC; AACGTT; AACGCT; AACGUC; AACGUU; AACGCU; AACGUT; AACGTU; GGCGTT; GGCGCT; GGCGTC; GGCGCC; GGCGUU; GGCGCU; GGCGUC; GGCGUT; GGCGTU.
  • the immunomodulatory polynucleotide comprises the sequence 5'-TGACTGTGAACGTTCGAGATGA-S' (SEQ ID NO:1).
  • the ISS comprises any of the sequences: 5'-TGACCGTGAACGTTCGAGATGA-S' (SEQ ID NO:2); 5 '-TCATCTCGAACGTTCCACAGTCA-S ' (SEQ ID NO.3); 5'-TGACTGTGAACGTTCCAGATGA-S' (SEQ ID NO:4); 5'-TCCATAACGTTCGCCTAACGTTCGTC-S' (SEQ ID NO:5); S'-TGACTGTGAABGTTCCAGATGA-S' (SEQ ID NO:6), where B is 5-bromocytosine; 5'-TGACTGTGAABGTTCGAGATGA-S ' (SEQ ID NO:7), where B is 5- bromocytosine and 5'-TGACTGTGAABGTTBGAGATGA-S' (SEQ ID NO:1)
  • An ISS and/or IS S -containing polynucleotide may contain modifications.
  • Modifications of ISS include any known in the art, but are not limited to, modifications of the 3'OH or 5'OH group, modifications of the nucleotide base, modifications of the sugar component, and modifications of the phosphate group. Various such modifications are described below.
  • An ISS may be single stranded or double stranded DNA, as well as single or double-stranded RNA or other modified polynucleotides.
  • An ISS may or may not include one or more palindromic regions, which may be present in the hexameric motif described above or may extend beyond the motif.
  • An ISS may comprise additional flanking sequences, some of which are described herein.
  • An ISS may contain naturally-occurring or modified, non-naturally occurring bases, and may contain modified sugar, phosphate, and/or termini.
  • phosphate modifications include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester and phosphorodithioate and may be used in any combination. Other non- phosphate linkages may also be used.
  • oligonucleotides of the present invention comprise phosphorothioate backbones.
  • Sugar modifications known in the field, such as 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs and 2'-alkoxy- or amino- RNA/DNA chimeras and others described herein, may also be made and combined with any phosphate modification.
  • Examples of base modifications include, but are not limited to, addition of an electron- withdrawing moiety to C-5 and/or C-6 of a cytosine of the ISS (e.g., 5-bromocytosine, 5-chlorocytosine, 5-fluorocytosine, 5-iodocytosine).
  • the ISS can be synthesized using techniques and nucleic acid synthesis equipment which are well known in the art including, but not limited to, enzymatic methods, chemical methods, and the degradation of larger oligonucleotide sequences. See, for example, Ausubel et al. (1987); and Sambrook et al. (1989).
  • the individual units When assembled enzymatically, the individual units can be ligated, for example, with a ligase such as T4 DNA or RNA ligase.
  • a ligase such as T4 DNA or RNA ligase.
  • Oligonucleotide degradation can be accomplished through the exposure of an oligonucleotide to a nuclease, as exemplified in U.S. Patent No. 4,650,675.
  • the ISS can also be isolated using conventional polynucleotide isolation procedures. Such procedures include, but are not limited to, hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences, antibody screening of expression libraries to detect shared structural features and synthesis of particular native sequences by the polymerase chain reaction.
  • Circular ISS can be isolated, synthesized through recombinant methods, or chemically synthesized. Where the circular ISS is obtained through isolation or through recombinant methods, the ISS will in some examples be a plasmid.
  • the chemical synthesis of smaller circular oligonucleotides can be performed using any method described in the literature. See, for instance, Gao et al. (1995) Nucleic Acids Res. 23:2025-2029; and Wang et al. (1994) Nucleic Acids Res. 22:2326-2333.
  • Naturally occurring DNA or RNA, containing phosphodiester linkages is generally synthesized by sequentially coupling the appropriate nucleoside phosphoramidite to the 5 '-hydroxy group of the growing oligonucleotide attached to a solid support at the 3 '-end, followed by oxidation of the intermediate phosphite triester to a phosphate triester.
  • the oligonucleotide is removed from the support, the phosphate triester groups are deprotected to phosphate diesters and the nucleoside bases are deprotected using aqueous ammonia or other bases.
  • the ISS can also contain phosphate-modified oligonucleotides. Synthesis of polynucleotides containing modified phosphate linkages or non-phosphate linkages is also know in the art. For a review, see Matteucci (1997) "Oligonucleotide Analogs: an
  • the phosphorous derivative (or modified phosphate group) which can be attached to the sugar or sugar analog moiety in the oligonucleotides of the present invention can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate or the like.
  • the preparation of the above-noted phosphate analogs, and their incorporation into nucleotides, modified nucleotides and oligonucleotides, per se, is also known and need not be described here in detail. Peyrottes et al.
  • Oligonucleotides with phosphorothioate backbones can be more immunogenic than those with phosphodiester backbones and appear to be more resistant to degradation after injection into the host.
  • ISS-containing polynucleotides used in the invention can comprise ribonucleotides (containing ribose as the only or principal sugar component), deoxyribonucleotides (containing deoxyribose as the principal sugar component), or, as is known in the art, modified sugars or sugar analogs can be incorporated in the ISS.
  • the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and a sugar "analog" cyclopentyl group.
  • the sugar can be in pyranosyl or in a furanosyl form.
  • the sugar moiety is in some examples the furanoside of ribose, deoxyribose, arabinose or 2'-0-alkylribose, and the sugar can be attached to the respective heterocyclic bases either in ⁇ or ⁇ anomeric configuration.
  • Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs and 2'-alkoxy- or amino-RNA/DNA chimeras.
  • heterocyclic bases, or nucleic acid bases, which are incorporated in the ISS can be the naturally-occurring principal purine and pyrimidine bases, (namely uracil or thymine, cytosine, adenine and guanine, as mentioned above), as well as naturally- occurring and synthetic modifications of said principal bases.
  • the ISS can include one or several heterocyclic bases other than the principal five base components of naturally-occurring nucleic acids.
  • the heterocyclic base in the ISS includes, but is not limited to, uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo [2.3-d] pyrrmidin-5-yl, 2-amino-4-oxopyrolo [2,3-d] pyrimidin-5-yl, 2-amino-4-oxopyrrolo [2.3-d] pyrimidin-3-yl groups, where the purines are attached to the sugar moiety of the ISS via the 9-position, the pyrrolidines via the 1 -position, the pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines via the 1 -position.
  • the ISS may comprise at least one modified base as described, for example, in the commonly owned US Pat. No. 6,562,798 (USSN 324,191) and international application WO 99/62923.
  • modified base is synonymous with “base analog”
  • modified cytosine is synonymous with “cytosine analog.”
  • nucleosides or nucleotides are herein defined as being synonymous with nucleoside or nucleotide “analogs.”
  • base modifications include, but are not limited to, addition of an electron-withdrawing moiety to C-5 and/or C-6 of a cytosine of the ISS.
  • the electron-withdrawing moiety is a halogen.
  • modified cytosines can include, but are not limited to, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5- fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodo uracil, 5-nitrocytosine, uracil, and any other pyrimidine analog or modified pyrimidine.
  • base-modified nucleosides and the synthesis of modified oligonucleotides using said base-modified nucleosides as precursors, has been described, for example, in U.S. Patents 4,910,300, 4,948,882, and 5,093,232. These base-modified nucleosides have been designed so that they can be incorporated by chemical synthesis into either terminal or internal positions of an oligonucleotide. Such base-modified nucleosides, present at either terminal or internal positions of an oligonucleotide, can serve as sites for attachment of a peptide or other antigen. Nucleosides modified in their sugar moiety have also been described (including, but not limited to, e.g., U.S. Patents 4,849,513, 5,015,733, 5,118,800, 5,118,802) and can be used similarly.
  • an ISS-containing polynucleotide is less than about any of the following lengths (in bases or base pairs): 10,000; 5,000; 2500; 2000; 1500; 1250; 1000; 750; 500; 300; 250; 200; 175; 150; 125; 100; 75; 50; 25; 10.
  • an ISS-containing polynucleotide is greater than about any of the following lengths (in bases or base pairs): 6; 7; 8; 10; 15; 20; 25; 30; 40; 50; 60; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 500; 750; 1000; 2000; 5000; 7500; 10000; 20000; 50000.
  • an ISS-containing polynucleotide is greater than about 6 bases or base pairs in length and less than about 200 bases or base pairs in length.
  • Conjugate molecule populations with varying subdural and immunomodulatory properties may be distinguished and/or defined by any of a number of structural and/or functional properties, including:
  • ratio of median mass of ISS-containing polynucleotide to median mass of antigen (d) ratio of median mass of ISS-containing polynucleotide to median mass of antigen; (e) ratio of (i) concentration of ISS-antigen conjugate required for inhibition of binding of antigen-specific antibody to antigen to (ii) concentration of antigen required for the same extent of inhibition of antigen-specific antibody to antigen (as discussed below, these ratios are usually, but need not be, calculated at 50% inhibition);
  • compositions comprising populations of structurally stable conjugate molecules, said conjugate molecules comprising an antigen and one or more polynucleotides comprising an immunostimulatory sequence (ISS), wherein said populations comprises any one or more of the properties described herein, either alone or in any combination.
  • the properties may be measured using standard techniques in the art and described herein, and it is understood that any of these properties may be measured in a variety of systems, including in vivo systems such as vertebrates and mammals, including, for example, mouse and/or human.
  • the "H" class is defined by any of the following properties, either alone or in any combination:
  • ratio of (i) concentration of ISS-antigen conjugate required for 50% inhibition of binding of antigen-specific antibody to antigen to (ii) concentration of antigen required for 50% inhibition of binding of antigen-specific antibody to antigen is about 3.5 to about 6.0 or more (including, but not limited to, 7.0, 8.0, 9.0, 10.0, 15, 20, 25, 30, 35, 40, 45, 50 or more) or alternatively is at least about any of the following: 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 9.0, 10, 15, 20, 25 (if expressed as a range, the upper limit may be any number, including those listed);
  • the ratio of (i) concentration of ISS-antigen conjugate required for 40% histamine release from basophils from an antigen-sensitized individual to (ii) concentration of antigen required for 40% histamine release from basophils from an antigen-sensitized individual is greater than about 300, greater than about 500, greater than about 750, greater than about 1000, greater than about 1250, greater than about 1400, greater than about 1500 (with an upper limit being any number, including, but not limited to, 750, 1000, 1250, 150O 5 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000);
  • ratio of (i) titers of total ThI- and Th2-associated antibodies elicited by ISS- antigen conjugate (in terms of antibodies elicited per unit mass of conjugate administered) to (ii) total ThI- and Th2-associated antibodies elicited by antigen (in terms of unit mass of antigen administered) is about or alternatively is less than about any of the following: 10, 7, 5, 4, 3.5, 3.0; 2.5, 2.0, 1.5, 1.0, 0.75, 0.5, 0.4, 0.3, 0.2, 0.1.
  • ratio of (i) titers of total ThI- and Th2-associated antibodies elicited by ISS- antigen conjugate (in terms of antibodies elicited per unit mass of conjugate administered) to (ii) total ThI- and Th2-associated antibodies elicited by antigen (in terms of 10 times the unit mass of antigen administered compared to amount conjugate administered) is about or alternatively is less than about any of the following: 1.0, 0.7, 0.6, 0.5, 0.4, 0.35; 0.3; 0.25, 0.2, 0.15, 0.11, 0.075, 0.05, 0.04, 0.03, 0.02, 0.01.
  • ratio of (i) titer of Thl-associated antibodies elicited by ISS-antigen conjugate (in terms of antibodies elicited per unit mass of conjugate administered) to (ii) titer of Thl-associated antibodies elicited by antigen (in terms of unit mass of antigen administered compared to amount conjugate administered) is about or alternatively is less than about any of the following: 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5;
  • ratio of (i) titer of Thl-associated antibodies elicited by ISS-antigen conjugate (in terms of antibodies elicited per unit mass of conjugate administered) to (ii) titer of Th2-associated antibodies elicited by conjugate about or alternatively is greater than about any of the following: 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10. If expressed as a range, the upper limit may be any number, including those listed, as well as others, such as 15, 20, 25, 30, 40, 50, 60, 75, 80, 90, 100;
  • the "M” class is defined by any of the following properties, either alone or in any combination:
  • ratio of (i) concentration of ISS-antigen conjugate required for 50% inhibition of binding of antigen-specific antibody to antigen to (ii) concentration of antigen required for 50% inhibition of antigen-specific antibody to antigen is about 2.5 to about 3.0 or alternatively about 3.25;
  • the ratio of (i) concentration of ISS-antigen conjugate required for 40% histamine release from basophils from an antigen-sensitized individual to (ii) concentration of antigen required for 40% histamine release from basophils from an antigen-sensitized individual is about 100 to about 200, or, alternatively, about 100, or alternatively, about between about 75 to about 250.
  • ratio of (i) titers of total ThI- and Th2-associated antibodies elicited by ISS- antigen conjugate (in terms of antibodies elicited per unit mass of conjugate administered) to (ii) total ThI- and Th2-associated antibodies elicited by antigen (in terms of unit mass of conjugate administered) is about 13 or alternatively is between about 10 or about 12 to about 100 (or, in some examples, about 12 to about 50);
  • ratio of titers of total ThI- and Th2-associated antibodies elicited by ISS- antigen conjugate in terms of antibodies elicited per unit mass of conjugate administered) to total ThI- and Th2-associated antibodies elicited by antigen (in terms of 10 times the unit mass of conjugate administered compared to amount conjugate administered) is about 1.3 or alternatively is between about 1.0 or about 1.20 to about 10 (or, in some examples, about 1.2 to about 5.0);
  • ratio of (i) titer of ThI -associated antibodies elicited by ISS-antigen conjugate in terms of antibodies elicited per unit mass of conjugate administered
  • titer of ThI -associated antibodies elicited by antigen in terms of unit mass of antigen administered compared to amount conjugate administered
  • the "L” class is defined by any of the following properties, either alone or in any combination: (a) an average of less than about 3 ISS-containing polynucleotides per antigen molecule;
  • ratio of (i) average mass of ISS-containing polynucleotide to (ii) average mass of antigen is (i) about 15 or alternatively less than about 15 (in some examples, about 10 or alternatively less than about 10) to (ii) about 40;
  • ratio of (i) concentration of ISS-antigen conjugate required for 50% inhibition of binding of antigen-specific antibody to antigen to (ii) concentration of antigen required for 50% inhibition of antigen-specific antibody to antigen is less than about 2.0, or alternatively is about 2.0;
  • the ratio of (i) concentration of ISS-antigen conjugate required for 40% histamine release from basophils from an antigen-sensitized individual to (ii) concentration of antigen required for 40% histamine release from basophils from an antigen-sensitized individual is less than about 75, or, alternatively, about 75 (in other examples, less than about 60 or alternatively about 60) to about 200, or, alternatively, to about 100;
  • ratio of (i) titers of total ThI- and Th2-associated antibodies elicited by ISS- antigen conjugate (in terms of antibodies elicited per unit mass of conjugate administered) to (ii) total ThI- and Th2-associated antibodies elicited by antigen (in terms of unit mass of conjugate administered) is about 150, or alternatively, greater than about any of the following: 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800. If expressed as a range, the upper limit may be any number, including the numbers listed.
  • ratio of (i) titers of total ThI- and Th2-associated antibodies elicited by ISS- antigen conjugate (in terms of antibodies elicited per unit mass of conjugate administered) to (ii) total ThI- and Th2-associated antibodies elicited by antigen (in terms of 10 times the unit mass of conjugate administered compared to amount conjugate administered) is about or alternatively is greater than about any of the following: 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80;
  • ratio of (i) titer of ThI -associated antibodies elicited by ISS-antigen conjugate (in terms of antibodies elicited per unit mass of conjugate administered) to (ii) titer of ThI -associated antibodies elicited by antigen is about 500 or more, including, but not limited to, about 500 or more, about 600 or more, about 700 or more, about 800 or more, about 900 or more, about 1000 or more.
  • ratio of (i) titer of ThI -associated antibodies elicited by ISS-antigen conjugate (in terms of antibodies elicited per unit mass of conjugate administered) to (ii) titer of Th2-associated antibodies elicited by conjugate is about or alternatively is less than about any of the following: 2.0, 1.5, 1.25.
  • conjugate populations characterized by any of the following (either alone or in any combination):
  • (a) ratio of (i) concentration of ISS-antigen conjugate required for 50% inhibition of binding of antigen-specific antibody to antigen to (ii) concentration of antigen required for 50% inhibition of antigen-specific antibody to antigen is any of more than about 1.5, 2.0, 2.25, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.50, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0. If expressed as a range, the upper limit may be any number, including those listed (for example, the conjugate population may be more than about 2.0, more than about 2.0 and less than about 5.5, more than about 2.0 and less than about 20.0).
  • ratio of (i) concentration of ISS-antigen conjugate required for 50% inhibition of binding of antigen-specific antibody to antigen to (ii) concentration of antigen required for 50% inhibition of antigen-specific antibody to antigen is any of less than about 1.5, 2.0, 2.25, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.50, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0. If expressed as a range, the lower limit may be any number listed as well as zero (for example, the conjugate population may be less than about 5.0, or alternatively less than about 5.0 and more than about 2.0).
  • the ratio of (i) concentration of ISS-antigen conjugate required for 40% histamine release from basophils from an antigen- sensitized individual to (ii) concentration of antigen required for 40% histamine release from basophils from an antigen-sensitized individual is at least about any of the following: 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 80, 90, 95, 100, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000.
  • the upper limit may be any number, including those listed. Alternatively, this ratio may be less than about any of the following: 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 80, 90, 95, 100, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000. If expressed as a range, the lower limit may be any number listed as well as zero.
  • ratio of antibody titer (more particularly, IgG titer, such as the sum of ThI- and Th2-associated IgG titer) elicited per unit mass of ISS-antigen conjugate to antibody titer (more particularly, IgG titer, such as the sum of ThI- and Th2-associated IgG titer) elicited per unit mass of antigen as at least more than about any of the following: 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2, 5, 10, 15, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 2000, 2250, 2500, 2750, 3000.
  • the upper limit may be any number, including those listed.
  • the ratio may be less than about any of the following: 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 2000, 2250, 2500, 2750, 3000.
  • the lower limit may be zero or any of the numbers listed.
  • ratio of ThI -associated antibody titer elicited by conjugate to ThI -associated antibody titer elicited by antigen is less than about any of the following: 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 150, 100, 50, 75, 60, 50, 40, 45, 30, 35, 25, 20, 14, 10, 5. If expressed as a range, the lower limit may be any number listed, including zero.
  • this ratio may be more than about any of the following: 10, 20, 50, 60, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 800, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 3000, 3500, 4000, 4500, 5000. If expressed as a range, the upper limit may be any number, including those listed.
  • the extent of conjugation can be controlled in a number of ways, all of which use chemical techniques well known in the art, which are also described herein.
  • One way to control extent of conjugation is to vary the equivalents of ISS in relation to linkage sites on antigen. That is, a constant amount or number of linkage sites is reacted with a particular amount of ISS.
  • the linkage sites could be controlled by, for example, choosing a certain linkage moiety that gave the desired number of linkage sites (for example, choosing to link via a carbohydrate as opposed to via amino groups), or alternatively, by controlling a linkage activating reaction such that the desired average number of linkage sites are activated.
  • a given antigen has a maximum number of potential linkage sites, depending on the nature of the antigen-ISS linkage. The extent of conjugation can be controlled by the number of these linkage sites which are used to link an ISS. Accordingly, the invention also includes examples in which the average percentage of total number of linkage sites attached to an ISS-containing polynucleotide is at least about any of the following: 5%, 10%, 20%, 30%, 33%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 97%, 98%.
  • the invention also includes examples in which the average percentage of total number of linkage sites attached to an ISS-containing polynucleotides is less than about any of the following: 10%, 20%, 30%, 33%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 97%, 98%.
  • the total number of linkage sites is determined by the mode of attachment. For example, if an antigen is linked to ISS-containing polynucleotide via a free amino group (such as in lysine), the total number of linkage sites is the number of lysines.
  • the total number of linkage sites is the total number of free sulfydryl groups. If antigen is linked via a carbohydrate moiety, then the total number of linkage sites is the total number of carbohydrate moieties. With respect to any of these examples, the average percentage of linkage sites attached to ISS-containing polynucleotide maybe accompanied by any of the immunomodulatory characteristics listed above, alone or in combination.
  • Conjugate molecule populations may be identified and/or characterized by any of a number of ways, including those listed above.
  • the extent of conjugation may be described by: (a) average or, alternatively median number of ISS to antigen molecules; (b) ratio of ISS to total linkage sites in antigen; (c) ratio of mass (whether average or median) of ISS to mass (whether average or median) of antigen; (d) ratio of ISS to T-cell epitopes in antigen; (e) ratio of ISS to B cell epitopes in antigen.
  • conjugate molecule populations of the invention may be characterized in terms of (a) degree of antigen-specific antibody response, such as IgG response; (b) ratio of ThI -associated antibodies to Th2-associated antibodies; (c) degree of suppression of histamine release; (d) degree of competition with antigen-specific antibody for binding to antigen; (e) degree of suppression of Th2-associated immune response; (f) secretion of ThI -associated cytokines, such as interferon; (g) secretion of Th2-associated cytokines, such as IL-4 and/or IL-5. Structural characterization
  • the extent of ISS-antigen conjugation may be determined using any number of protein and nucleic acid measurement methods known in the art.
  • antigen and/or protein-specific detection techniques for example, antigen-specific antibodies and/or Coomassie Blue stain
  • nucleic acid-specific detection techniques for example, hybridization with detectably-labeled DNA probes
  • the amount of polynucleotide to antigen may be determined.
  • the amount of oligonucleotide bound to a polypeptide may also be determined by the measurement of size or molecular weight of the conjugate molecule.
  • Conjugate molecule size may be determined using methods known in the art including, but not limited to, sodium dodecylsulfate polyacrylamide electrophoresis (SDS-PAGE) analysis and size- exclusion chromatography (SEC).
  • the ISS-antigen conjugate molecules may be analyzed using a combination of size determination and/or separation techniques and nucleic acid and protein determination techniques. For example, after fractionation of conjugate molecule reaction products using SEC, the protein and nucleic acid content of each fraction may be determined by the absorbance of the fraction at 280 ran and 260 nm, respectively. In this way, the results of both the size of the conjugate molecule and the nucleic acid and protein detection analysis may be combined to characterize the structure of the conjugate molecule. The ratio of the amount of polynucleotide to the amount of protein in each conjugate molecule fraction indicates the average number of ISS molecules per antigen molecule.
  • antigen-specificity and antibody class and/or subclass of the antibodies generated in response to administration of ISS-antigen conjugate molecules may be determined using standard ELISA format assays.
  • assays for example, antigen is attached to a substrate and incubated with serum from a ISS-antigen conjugate molecule treated individual. The amount of antigen-specific antibody attached to the substrate-bound antigen is then determined using antibody-specific reagents, such as antibodies specific for IgGl, IgG2, IgG3, IgG4, IgE, etc.
  • Methods known in the art may be used to determine a concentration of ISS- antigen conjugate molecule required for inhibition of binding of antigen-specific antibodies to antigen, such as competitive ELISA assays as described herein.
  • Methods known in the art may be used to measure the amount of histamine release from basophils from an antigen-sensitized individual in response to ISS-antigen conjugate molecule.
  • the amount of histamine released into the cell culture supernatant may be determined after leukocytes from blood of allergic individuals are treated with varying concentrations and/or preparations of ISS-allergen conjugate molecules.
  • Methods known in the art may be used to determine the cytokine production profiles generated in response to administration of ISS-antigen conjugate molecules. For example, the supernatants of cells treated with ISS-conjugate molecules in vitro are analyzed for the presence of cytokines. The types and amounts of cytokines produced by lymphocytes exposed to ISS-antigen conjugate molecules may be measured using standard ELISA format assays.
  • a cytokine profile produced in response to an ISS-antigen conjugate molecule may also be determined using standard cytokine bioassays including, but not limited to, those in which cell survival is dependent on the presence of a particular cytokine (for example, IL-2) and those in which a particular cytokine (for example, interferon) inhibits viral replication.
  • a particular cytokine for example, IL-2
  • a particular cytokine for example, interferon
  • a class of conjugate molecule may also be characterized by the extent of antigen-specific antibody suppression after administration or relative to administration of antigen alone. For example, levels of serum antibodies may be determined before and after administration of the ISS-antigen conjugate molecule and/or antigen alone. The antibody levels at various time points may then be compared to determine the extent of antibody suppression.
  • a class of conjugate molecule may also be characterized by the extent of antibody response, and in some examples, an antigen-specific antibody response, especially an IgG response. As noted above, a class may be characterized by a ratio of (i) IgG antibodies produced in response to conjugate molecule to (ii) IgG antibodies produced in response to antigen alone. For these characterizations and examples, the ratio may be
  • Th2-associated antibody or antibodies elicited by ISS-antigen conjugate molecule to (ii) Th2-associated antibodies elicited by antigen.
  • a Thl-associated antibody is an antibody associated with a ThI response.
  • IgG2a is associated with a ThI response.
  • IgGl and/or IgG3 antibodies appear to be associated with a ThI response. See, e.g., Widhe et al. (1998)
  • Th2-associated antibody is an antibody associated with a Th2 response.
  • IgGl is associated with a Th2 response.
  • IgG2 and/or IgG4 appear to be associated with a Th2 response (Widhe et al. (1998) and de Martino et al. (1999)).
  • IgE is associated with a Th2 response. It is understood that, for these characterizations and examples, any one or more type of antibody may be evaluated, as long as the same antibody or antibody production level is compared to that elicited by antigen alone.
  • the unit mass of the conjugate molecule may be in terms of mass of antigen component of conjugate molecule, polynucleotide component of conjugate molecule, and/or mass of conjugate molecule. For example, if a conjugate molecule has a total molecular weight of 100, with the antigen component accounting for 80 and the ISS component accounting for 20, the unit mass for purposes of calculating and comparing levels of antibody production may be any of 100, 80, or 20.
  • the Examples provide calculations in which the mass of the antigen component of the conjugate molecule (Amb a 1) serves as the basis for calculating and comparing levels of antigen production compared to antigen alone.
  • mass of conjugate molecule to mass of antigen may or may not be 1 : 1.
  • antibody produced by unit mass of conjugate molecule is compared to antibody produced by any of 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 40 times the mass of antigen.
  • Amb a 1 antibody produced by 1 ⁇ g of conjugate molecule (as measured by the amount of antigen; thus 1 ⁇ g of antigen in the conjugate molecule) is compared to antibody produced by 10 ⁇ g of Amb a 1.
  • the ISS-containing polynucleotide is conjugated with the antigen.
  • the ISS portion can be coupled with the antigen portion of a conjugate molecule in a variety of ways, including covalent and/or non-covalent interactions.
  • the link between the portions can be made at the 3' or 5' end of the ISS, or at a suitably modified base at an internal position in the ISS.
  • the antigen is a peptide and contains a suitable reactive group ⁇ e.g., an N-hydroxysuccinimide ester) it can be reacted directly with the N 4 amino group of cytosine residues. Depending on the number and location of cytosine residues in the ISS, specific coupling at one or more residues can be achieved.
  • modified oligonucleosides such as are known in the art, can be incorporated at either terminus, or at internal positions in the ISS. These can contain blocked functional groups which, when deblocked, are reactive with a variety of functional groups which can be present on, or attached to, the antigen of interest.
  • this portion of the conjugate molecule can be attached to the 3 '-end of the ISS through solid support chemistry.
  • the ISS portion can be added to a polypeptide portion that has been pre-synthesized on a support. Haralambidis et al. (1990a) Nucleic Acids Res. 18:493-499; and Haralambidis et al. (1990b) Nucleic Acids Res. 18:501-505.
  • the ISS can be synthesized such that it is connected to a solid support through a cleavable linker extending from the 3 '-end.
  • the peptide portion of the conjugate molecule can be attached to the 5 '-end of the ISS through an amine, thiol, or carboxyl group that has been incorporated into the oligonucleotide during its synthesis.
  • a linking group comprising a protected amine, thiol, or carboxyl at one end, and a phosphoramidite at the other, is covalently attached to the 5'-hydroxyl.
  • An ISS-antigen conjugate molecule can also be formed through non-covalent interactions, such as ionic bonds, hydrophobic interactions, hydrogen bonds and/or van der Waals attractions.
  • Non-covalently linked conjugate molecules can include a non-covalent interaction such as a biotin-streptavidin complex.
  • a biotinyl group can be attached, for example, to a modified base of an ISS. Roget et al. (1989) Nucleic Acids Res. 17:7643- 7651. Incorporation of a streptavidin moiety into the peptide portion allows formation of a non-covalently bound complex of the streptavidin conjugated peptide and the biotinylated oligonucleotide.
  • Non-covalent associations can also occur through ionic interactions involving an ISS and residues within the antigen, such as charged amino acids, or through the use of a linker portion comprising charged residues that can interact with both the oligonucleotide and the antigen.
  • non-covalent conjugation can occur between a generally negatively-charged ISS and positively-charged amino acid residues of a peptide, e.g., polylysine, polyarginine and polyhistidine residues.
  • Non-covalent conjugation between ISS and antigens can occur through DNA binding motifs of molecules that interact with DNA as their natural ligands. For example, such DNA binding motifs can be found in transcription factors and anti-DNA antibodies.
  • the linkage of the ISS to a lipid can be formed using standard methods. These methods include, but are not limited to, the synthesis of oligonucleotide-phospholipid conjugate molecules (Yanagawa et al. (1988) Nucleic Acids Symp. Ser. 19:189-192), oligonucleotide-fatty acid conjugate molecules (Grabarek et al. (1990) Anal. Biochem. 185:131-135; and Staros et al. (1986) Anal. Biochem. 156:220-222), and oligonucleotide- sterol conjugate molecules. Boujrad et al. (1993) Proc. Natl. Acad. Sd. USA 90:5728- 5731.
  • the linkage of the oligonucleotide to an oligosaccharide can be formed using standard known methods. These methods include, but are not limited to, the synthesis of oligonucleotide-oligosaccharide conjugate molecules, wherein the oligosaccharide is a moiety of an immunoglobulin. O'Shannessy et al. (1985) J. Applied Biochem. 7:347-355.
  • the linkage of a circular ISS to a peptide or antigen can be formed in several ways. Where the circular ISS is synthesized using recombinant or chemical methods, a modified nucleoside is suitable. Ruth (1991), pp.
  • kits comprising a composition comprising a structurally stable conjugate molecule and any one or more additional components as described herein, including but not limited to a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0; an amino acid, a carbohydrate, a surfactant, and/or other pharmaceutically acceptable carrier.
  • a kit may comprise an article of manufacture containing such compositions, wherein the composition may be in liquid or lyophilized form.
  • an article of manufacture comprises a composition comprising a conjugate molecule wherein the composition comprises greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95% or greater than about 97% of the conjugate molecule in non-aggregate form at a temperature of between about 2 degrees C and about 8 degrees C.
  • a kit may comprise at least 2, at least 3, at least 4, at least 5 or at least 6 articles of manufacture comprising said composition.
  • the allergen is Amb a 1.
  • aggregation is measured by RALS.
  • an article of manufacture comprises a liquid composition comprising a conjugate molecule and in other examples a lyophilized composition comprising a conjugate molecule.
  • an article of manufacture comprises a reconstituted liquid composition (that is, reconstituted from a lyophilized composition) comprising a conjugate partner.
  • the kits of the invention may optionally contain instructions for their use (for example, instructions for any of the methods described herein) and/or any other suitable components.
  • compositions comprising a structurally stable conjugate molecule as described herein are especially useful for administering to an individual in need of immune modulation (in the context of, for example, infectious disease, cancer, and/or allergy).
  • the present invention provides pharmaceutical compositions comprising a structurally stable conjugate molecule.
  • Methods generally comprise administration of a composition comprising a structurally stable conjugate molecule or conjugate molecule population as described herein to an individual in an amount sufficient to modulate an immune response in the individual.
  • methods of modulating an immune response comprise administering a composition comprising a conjugate molecule such that the desired modulation of the immune response is achieved. Assessment of immune responses have been described above.
  • a kit comprises an article of manufacture comprising a composition comprising structurally stable AIC in a range of about 0.1 ⁇ .g to about 200 ⁇ g.
  • a composition comprises AIC in a range of at least about lO ⁇ g, 20 ⁇ g, 30 ⁇ g, 4O j Ug 3 50 ⁇ g, 60 ⁇ g, 70/xg, 80 ⁇ g, 90 ⁇ g or lOO ⁇ g and up to about 110/ig, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, 150 ⁇ g, 160 ⁇ g, 170 ⁇ g, 180 ⁇ g, 190 ⁇ g or 200 ⁇ g.
  • a composition comprises AIC in a range of between about 30 ⁇ g and about 60 ⁇ g.
  • the conjugate molecule comprises an allergen, and in other examples, the conjugate molecule comprises Amb a 1.
  • the conjugate molecule is AIC.
  • the composition comprises AIC and a component capable of maintaining the pH of the composition in the range of about 6.0 to about 9.0 and additionally may contain any one or more of the following; 1) an amino acid; 2) a carbohydrate; 3) a surfactant, or 4) other pharmaceutically acceptable carrier as long as the AIC is structurally stable within the composition.
  • the invention provides methods of modulating an immune response in an individual which comprise administering a composition comprising any of the conjugate molecules or conjugate molecule populations described herein to the individual in an amount sufficient to modulate the immune response.
  • the individual is in need of, or will be in need of, such modulation, due, for example, for a disease condition or being at risk of developing a disease condition.
  • disease conditions include, but are not limited to, allergy, cancer, infectious diseases (such as viral or bacterial infection).
  • the disease condition is an allergy.
  • the immune modulation comprises stimulating a (i.e., one or more) ThI -associated cytokine, such as interferon- ⁇ .
  • the immune modulation comprises suppressing production of a (i.e., one or more) Th2-associated cytokine, such as IL-4 and/or IL-5. Measuring these parameters uses methods standard in the art and has been discussed above.
  • one (or more) ThI -associated cytokines is produced, while antigen-specific antibody production is suppressed. Measuring these parameters uses methods standard in the art and has been discussed above.
  • the immune modulation comprises stimulating a (i.e., one or more) ThI -associated cytokine, such as interferon- ⁇ , and suppressing production of antigen-specific antibodies.
  • ThI -associated cytokine such as interferon- ⁇
  • the immune modulation comprises suppression of histamine release. Degrees of suppression of histamine release for various conjugate molecule populations have been described above and apply to these methods.
  • methods of suppressing antibody formation, preferably antigen-specific antibody formation, in an individual, while stimulating production of a ThI -associated cytokine comprise administering a composition comprising a population ISS-antigen conjugate molecules of the H class to the individual whereby antibody formation is suppressed while a ThI -associated cytokine is stimulated. Measuring these parameters uses methods standard in the art and has been discussed above.
  • the invention provides methods of treating an allergic condition in an individual which comprise administering any of the compositions comprising a structurally stable conjugate molecule or conjugate molecule population as described herein in which the antigen is an allergen in an amount sufficient to ameliorate or palliate the allergic condition, generally by modulating the immune response to the antigen.
  • Palliation can be determined by, for example, alleviation of one or more symptoms associated with allergy.
  • the invention also provides methods of reducing allergenicity of an antigen, particularly an allergen, comprising administering a composition comprising a structurally stable conjugate molecule as described herein to an individual in need, such that allergenicity is reduced.
  • the conjugate molecule is AIC.
  • Routes of administration include, but are not limited to, intravascular, arterial or venous; subcutaneous; intraperitoneal; intraorganal; intramuscular; oral; transmucosal; epidermal; parenteral; and gastrointestinal or the like.
  • the compounds may be provided in the medium of the cells and/or organ, as a single bolus, by repetitive addition, by continual infusion, or the like.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association a conjugate molecule or conjugate molecule population with components as described herein. To determine the optimum concentration of conjugate molecule for any application, conventional techniques may be employed.
  • Compositions may include aqueous and non-aqueous isotonic sterile injection solutions which may contain bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient as long as the conjugate molecule remains structurally stable within the composition. If a composition comprising a structurally stable conjugate molecules is provided as an aerosol, propellant(s) known in the art may be added to a liquid composition.
  • propellant(s) known in the art may be added to a liquid composition.
  • the invention also provides methods of making compositions comprising structurally stable conjugate molecules comprising any of the techniques and/or steps described herein. Accordingly, provided herein are methods for preparing a composition comprising a structurally stable conjugate molecule comprising combining a conjugate molecule with a component capable of maintaining the pH in the range of about 6.0 to about 9.0. Such components are described herein.
  • the method of preparing a composition comprising a structurally stable conjugate molecule further comprises the step of combining the conjugate molecule with any one or more of 1) an amino acid as described herein; 2) a carbohydrate as described herein; 3) a surfactant; and 4) a pharmaceutically acceptable carrier in any order, as long as the conjugate molecule remains structurally stable.
  • the present invention also provides methods of preparing a lyophilized composition comprising the step of lyophilizing a liquid composition comprising a structurally stable conjugate molecule.
  • the present invention also provides methods of preparing a reconstituted composition comprising reconstituting a lyophilized composition.
  • the Amb a 1-ISS Oligonucleotide Conjugate is a protein-oligonucleotide conjugate prepared by covalently linking the purified short ragweed antigen Amb a 1 to a phosphorothioate immunostimulatory (ISS) oligonucleotide.
  • the Amb a 1 is isolated from the pollen at 2-8 0 C by extraction and ammonium sulfate precipitation.
  • the crude extract is processed at ambient temperature through two chromatography steps.
  • Amb a 1 is purified by DEAE Sepharose Fast Flow anion exchange chromatography followed by Butyl Sepharose Fast Flow hydrophobic interaction chromatography.
  • Crosslinking is via the heterobifunctional linker sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane-l-carboxylate (sSMCC).
  • sSMCC creates stable amide and thioether bonds between the ISS oligonucleotide and the Amb a 1.
  • AIC has an average of approximately 4.0 moles of ISS oligonucleotide per mole of Amb a 1 and has an average molecular weight of approximately 65 IcDa.
  • the Amb a 1 antigen (molecular weight of approximately 37,800 Da) is purified from defatted short ragweed pollen (Ambrosia artemisiifolia) using standard chemical and chromatographic techniques.
  • the activated Amb a 1 maleimide group reacts with the sulfhydryl of the 5' Thio ISS oligonucleotide to form a stable thioether bond, covalently linking the Amb a 1 to the ISS and creating AIC.
  • AIC is purified by Superdex HR 200 Gel Filtration chromatograph.
  • the 5 'Disulfide ISS oligonucleotide which contains the immunostimulatory hexamer motif 5'-AACGTT, has the sequence S'-Disulfide-TGACTGTGAACGTTCGAGATGA-S'.
  • the theoretical molecular weight is approximately 7500 Da.
  • AIC-L, AIC-M 3 and AIC-H are covalent conjugate molecules of the ragweed allergen Amb a 1 and the IS S -containing polynucleotide 5'- TGACTGTGAACGTTCGAGATGA-S ' (SEQ ID NO:1). All three classes of conjugate molecule are prepared from the same ISS-containing polynucleotide and employing the same heterobifunctional linker. The number of oligonucleotides conjugated to the Amb a 1 can distinguish the classes. The amount of oligonucleotide bound to Amb a 1 can be determined by the measurement of size or the molecular weight of the conjugate molecules.
  • AIC-L contains an average of 2-3 oligonucleotides per Amb a 1 molecule
  • AIC-M an average of 3.5 to 4.5
  • AIC-H contains an average of >5.5.
  • the 5 'disulfide ISS oligonucleotide was synthesized as a phosphorothioate on a controlled pore glass support (CPG) using an automated synthesizer.
  • the required sequence was assembled using the standard ⁇ -cyanoethylphosphoramidite 'DMT off approach of detritylation, coupling, oxidation, and capping.
  • the HSP process produces lyophilized 5'- Disulfide ISS oligonucleotide as a bulk pharmaceutical that is released by HSP.
  • Triscarboxyethylphosphine (TCEP) was allowed to reach ambient temperature and dissolved in 10 mM NaPO 4 ZMl mM NaCl/pH 7.2.
  • the 5 ' disulfide ISS oligonucleotide was allowed to reach ambient temperature, dissolved in the same buffer, and treated with the TCEP solution for 2 hours at 40 °C. This material was carried on directly to the isolation step.
  • NEM N-ethyl maleimide
  • DMSO dimethyl sulfoxide
  • Amb a 1 was thawed and treated with the NEM solution for 2 hours at 2O 0 C.
  • Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC) was allowed to reach ambient temperature and dissolved in DMSO.
  • the NEM blocked Arab a 1 was treated with the sSMCC solution for 2.5 hours at 20 0 C. This material was carried on directly to the isolation step.
  • the crude AIC-L conjugate molecule was prepared by incubation of a mixture of 4 molar equivalents of 5' thio ISS oligonucleotide and 1 molar equivalent the maleimide activated Amb a 1 for 3 hours at 20 °C.
  • Crude AIC-M and AIC-H were prepared in a similar manner but by addition of 7 and 17 molar equivalents of 5' thio ISS oligonucleotide, respectively.
  • a pre-packed gel filtration column was equilibrated with 10 mM NaPO 4 /! 41 mM NaCl/pH 7.2 buffer and the crude AIC-L, AIC-M, or AIC-H were loaded onto the column.
  • the AIC was eluted isocratically with 10 mM NaPO 4 /141 mM NaCl/pH 7.2 buffer.
  • Example 2 Stability Profiling of Compositions Comprising AIC
  • AIC in PBS the AIC was prepared by dilution of AIC into a 1OmM sodium phosphate, 14ImM sodium chloride pH 7.2 (PBS) composition followed by a sterile filtration and was stored frozen at ⁇ -60°C.
  • PBS 14ImM sodium chloride pH 7.2
  • Real-time stability studies on the AIC in PBS demonstrated at least 24 months stability when stored at ⁇ -60°C.
  • the ⁇ -60°C storage temperature was chosen because accelerated stability studies revealed that AIC drug product underwent aggregation with time when stored liquid at 2-8 0 C in PBS. This aggregation resulted in an approximately 30% decrease in AIC monomer content in 12 months as evaluated by a Size Exclusion Chromatography (SEC) method.
  • SEC Size Exclusion Chromatography
  • Stability profiling on liquid compositions comprising AIC revealed that negatively charged components destabilized AIC resulting in a presumed conformational change and aggregation.
  • the sodium phosphate and sodium chloride of the PBS formulation both contributed to the aggregation of AIC.
  • Additional stability profiling resulted in the development of an AIC composition containing 2OmM histidine, 5OmM glycine, 21OmM sucrose, pH 7.5 (HGS) that was structurally stable as a liquid at 2-8°C. Accelerated studies on AIC in HGS showed minimal loss of AIC monomer at 20-22°C when compared to the AIC in PBS composition. Results using the SEC method for AIC stored at both ⁇ -60°C and 2-8 0 C are shown in Tables 4 and 5 shown below.
  • Example 3 Activity of AIC in PBS or HGS: Murine IgG2a activity
  • AIC The biological activity of AIC was determined by the ability to generate an Amb a 1 specific IgG2a response in BALB/c mice. Twelve-week-old female BALB/c mice from Jackson Labs were immunized intradermally in the tail twice at two-week intervals with 1 ⁇ g doses of AIC (10 mice/group). Amb a 1 specific IgG2a titers were determined by ELISA from serum collected two weeks post 2 nd immunization. Nunc Maxisorp 96-well plates were coated with 1 ⁇ g/ml Amb a 1 in phosphate buffer overnight at 4°C, washed, and blocked. Serum dilutions were loaded on plates and incubated at 4°C overnight.
  • Amb a 1 IgG2a antibodies were detected with a biotinylated goat anti-mouse IgG2a conjugate molecule. After treatment with a streptavidin - HRP conjugate molecule plates were developed with 3,3',5,5' tetramethylbenzidine. A 450 was determined on an ELISA plate reader. Titers were calculated as the reciprocal of the serum dilution that gives an A 450 of 0.5.
  • Example 4 Allergenicity of AIC in PBS or HGS: Histamine Release
  • in vitro characterization of AIC in HGS and PBS was performed. Specifically, the allergenicity of these compositions were tested using in vitro histamine release assay. The allergenicity of AIC in PBS and HGS was compared to Amb a 1 using an in vitro histamine release assay. In this assay, leukocytes were prepared from blood of ragweed allergic patients. These cells were incubated for 45 minutes with concentrations of Amb a 1 or AIC ranging from 0.0001 to 1.0 ⁇ g/ml. The cells were then pelleted by centrifugation and the supernatants were analyzed for histamine content by automated fmorometry.
  • the inventors discovered that the structural stability of a conjugate molecule is dependent upon temperature, salt and pH conditions.
  • the present inventors have found that a conjugate molecule comprising an antigen undergoes aggregation with time when stored liquid at 2-8 degrees C in compositions comprising sodium phosphate and sodium chloride. Without being bound by theory, it is believed that due to the negative charge of the conjugate molecule, by virtue of the presence of an ISS, compositions comprising non- negatively charged components or components having a neutral charge or non-polar components are desired to maintain the structural stability of the conjugate molecule present in the composition.
  • the structural stability of a conjugate molecule comprising the allergen Amb a 1 within various composition was characterized using IF, EF RALS, HPLC-SEC, SDS PAGE as described herein.
  • IF Intrinsic Fluorescence
  • pure Tryptophan has an emission max of 355 ran in a polar environment and 305 nm in a non- polar environment. Therefore, a ratio of intensities (high/low) can yield information about the unfolding/structural response to external stimuli. Further, by using a ratio, inaccuracies caused by differences in AIC concentration and changes in instrumentation are eliminated. In IF, the higher the ratio, the more unfolded the molecule (the Trp residues are exposed to a more polar environment).
  • Extrinsic Fluorescence utilizes an external, non-covalent, polarity- sensitive fluorescent probe, such as for example, ANS (8-anilino-naphthalene sulphonic acid), to probe a conjugate molecule's apparent exposure of hydrophobic domains and to monitor possible changes in this parameter as a function of various environmental stresses and conditions.
  • Fluorescent probes that have an affinity for hydrophobic domains on conjugate molecules can complement intrinsic fluorescence. Changes in AIC or other conjugate molecules induced by various solution stresses can result in changes in the hydrophobic domains to which such probes bind, which in turn can affect the spectral characteristics of the non-covalently bound probe fluorescence.
  • ANS fluorescence in the absence of AIC is independent of pH and temperature.
  • ANS has an emission max of 520 nm in a polar environment and 490 nm in a non-polar environment. Therefore, a ratio of fluorescent emissions at 520 nm and 490 nm (520/490) is indicative of the apparent exposure of hydrophobic domains on AIC or other conjugate molecules examined, with a lower ratio indicating increasing hydrophobic cleft availability to the probe, and therefore more unfolding of the molecule.
  • Right Angle Light Scatter can be employed to detect and monitor the subtle changes in associative behavior of stressed conjugate molecules in otherwise visually clear formulations. Conformational changes in conjugate molecules can result in an association between molecules to possibly exclude polar water. This associative behavior can be subtle or can be easily detected as soluble and insoluble aggregates or even visible precipitation. RALS monitors macroscopic changes in an otherwise soluble molecule transitioning to insoluble aggregates. On a scale of 0-10 (instrument scale of minimum/maximum intensity), 10 could suggest self-association in otherwise visually clear formulations.
  • the structural stability of most conjugates is influenced by pH.
  • AIC was probed for possible pH-induced conformational changes during pH-transitions using RALS, IF, and EF. This was performed by dialyzing AIC into the Base Buffer (as described below) that allows for easy pH transitions and then titrating with HCl and NaOH while observing RALS.
  • product stability was examined using a range of pH conditions, using IF and RALS or EF.
  • a 30 pg/mL AIC solution was prepared in 10 mM Sodium Phosphate, 141.7 mM NaCl at pH 7.2 (PBS). An absorbance scan was run on this sample using a UV spectrophotometer. The wavelength of maximum absorbance was found to be 258 nm. The sample was then examined using excitation and emission scans. Although AIC exhibited a maximal absorbance at 258 run (that is, 258nm), the IF excitation was chosen at 295 nm because the excitation of T ⁇ overlaps that of Phe and Tyr. The IF excitation wavelength was therefore chosen 10 nm above the maximum. A ratio of emissions wavelength is used to assess changes in conformation. The emission wavelengths of 295 nm were chosen based on an emissions scan, with the wavelengths that yield a half- maximal emission value being selected. For AIC, these wavelengths were 323 nm and 363 nm.
  • RALS and EF do not require wavelength determinations. However, RALS does require a voltage determination, and the voltage used varies according to AIC concentration. RALS is best monitored by setting excitation and emission wavelengths at 320 nm.
  • the extrinsic probe ANS is excited at 380 nm and the emissions observed at 490 nm and 520 nm.
  • ANS has an emission max of 520 nm in a polar environment and 490 nm in a non-polar environment. Therefore, a ratio of fluorescent emissions at 520 nm and 490 nm (520/490) is indicative of the apparent exposure of hydrophobic domains on AIC examined, with a lower ratio indicating increasing hydrophobic cleft availability to the probe.
  • ANS 8-anilino-naphthalene sulphonic acid
  • ANS was titrated into a 400 pL- sample of drug (30 ⁇ g/mL) in its current formulation.
  • a final concentration for ANS obtained by adding 7 AL of a 10 mM stock to 400 gL of 30 pg/mL sample, was determined as most appropriate for tests.
  • Guanidine titration was used to determine the maximum possible IF intensity changes resulting from changes in the Trp environment due to conformational changes.
  • Samples were prepared containing Guanidine ranging from 0 M to 6 M (incubated at room temperature for 30 minutes), and fluorescence emission scans were performed. The scans were obtained using 295 nm Excitation. Three ratios using emission wavelengths at the maximum emission wavelength (343) and at +20 nm and -20 nm from the peak.
  • AIC appeared to be moderately shear stress sensitive.
  • the RALS increased substantially at 3 hours, after which no further increase in RALS was observed.
  • short-term shearing should have minimal or no effect. Note that at higher concentrations this sensitivity could become a substantial issue.
  • NaCl, pH 7.2 does not undergo aggregation and precipitation in response to increasing temperature. Repeated cycles of freeze-thaw do not appear to alter the molecule's response to a temperature ramp, since there was no increase in RALS as the sample were heated.
  • the IF ratio initially decreased as the temperature increased, suggesting conformational changes associated as a function of temperature, resulting in increasing the Trp residues' exposure to the buffer.
  • the molecule undergoes a substantial conformational change, suggested by the increase in IF ratio.
  • This conformational change is not accompanied by a detectable aggregation, either visually in the fluorimeter cuvette or by RALS measurement.
  • the external, non-covalent, polarity-sensitive fluorescent probe, ANS has an emission max of 520 nm in a polar environment and 490 nm in a non-polar environment. Therefore, a ratio of 520/490 is indicative of the apparent hydrophobicity of AIC solution examined. A higher ratio indicates that the molecule is more hydropliilic and a lower ratio indicates that it is exposing hydrophobic domains for ANS binding.
  • PyMaI Assay PyMaI [N- (1-pyrene) maleimide] solubilized in DMSO (Dimethyl Sulfoxide) is an extrinsic -SH specific probe used to detect the possible presence of free -SH groups in the molecule.
  • DMSO Dimethyl Sulfoxide
  • PyMaI reacts with a free sulfhydryl group it becomes fluorescent, with peak fluorescence at 374 nm and 394 nm.
  • a 20-fold molar excess of PyMaI was added to a sample of AIC at 30 ⁇ g/mL and samples incubated for 30 minutes in the presence of either 6M Guanidine or 10% SDS to unfold the molecule.
  • the data has buffer samples subtracted from the fluorescent values. Data suggest that there are no free sulthydryls groups concealed within the molecule in its native state, since the PyMaI fluorescence was identical when comparing the native PBS and SDS-pretreated samples. Guanidine appeared to suppress the reaction of PyMaI with AIC. Since the PyMaI fluorescence increased over time, the data also suggested that the molecule potentially has free sulfhydryl groups exposed even in its native state that could potentially result in aggregation. The data do not provide quantifiable measurements of free sulfhydryls.
  • BB Base Buffer
  • BB is a multi-buffer system designed to facilitate easy pH transitions while using a minimal concentration of buffer to reduce stability affects from the buffer itself.
  • a lOX-strength solution is prepared as follows:
  • the 1OX BB is diluted to 1 X (resulting in a final concentration of 2 mM for each buffer component), desired components added, and pH is adjusted as required.
  • each BB buffer was prepared with varying concentrations of NaCl (OmM, 1OmM, 10OmM, 50OmM) and the solution adjusted to pH 7.2 in order to match the PBS composition.
  • AIC (about 300 ⁇ g/condition) was dialyzed in Slide- A-Lyzer dialysis cassettes against BB with varying NaCl concentrations. The dialysis buffer was changed three times, with the second incubation occurring overnight.
  • samples were diluted and then examined by UV Spectrophotometry to determine the concentration. Subsequently, the samples were further diluted to yield the final desired concentration of 30 ⁇ g/mL.
  • samples were sterilized using 0.2 gm PES filters into 15 mL sterile tubes, and then aliquoted into 1.5 mL sterile cryovials (polypropylene, Coming cat. no. 430659). These samples were stored at 4°C until the next morning, where they were placed in storage boxes in incubators.
  • two sets of about 1.1 mL samples were placed at 30°C, and one set was placed at 4O 0 C. The remaining sample set was stored at 4°C until the samples were analyzed by EF, IF; RALS and by pH Titration (see below).
  • the ionic strength samples presented here were also incubated at 30°C and 4O 0 C for 7 days (t 7 ) and analyzed by RALS, IF, and EF. Aliquots from these samples were frozen for later examination by SEC-HPLC.
  • the 40°C 7 day incubation looks nearly identical to the results obtained from the 3O 0 C incubation.
  • the only difference is a small increase in RALS in the 100 mM and 500 mM NaCl conditions in samples incubated at 4O 0 C (Fig. 1). This suggests that this higher temperature may reveal subtle differences in AIC behavior as examined with RALS.
  • Similar results were obtained in the same formulations incubated for 14 days at 30 0 C.
  • the 500 mM NaCl formulation exhibited a substantial increase in aggregation as determined by RALS.
  • Trp Tryptophans
  • AIC exhibits a conformational change with a Tm of about 50°C, which is the same for all compositions.
  • the 500 mM NaCl sample exhibits the greatest conformational change, followed by the 100 mM sample.
  • the composition which contains 141 mM NaCl, exhibits a conformational change equivalent to that observed for the 100 mM sample, as judged by the IF ratio.
  • the stability of AIC was examined by IF using the t7 samples.
  • the t7 samples indicate that at lower ionic strength conditions, the AIC molecule exhibits conformation changes indicative of unfolding (based on the Guanidine
  • AU formulations display the same conformational changes (IF ratio changes at the same Tm) as the temperature increases, but the lower IF ratios in the lower ionic strength conditions indicate that AICs Trp residues are more exposed to a polar environment.
  • the samples incubated for 7 days at 40°C did not exhibit the dramatic conformational changes at elevated temperatures observed in samples incubated at 30°C.
  • the 500 mM NaCl formulation exhibited a substantial change in conformation as determined by IF.
  • Ionic strength samples were temperature-ramped and examined using extrinsic fluorescence.
  • % non-aggregation was substantially reduced (that is, % aggregation increased) at high NaCl concentration (500 mm).
  • the 100 MM NaCl sample exhibited a slight decrease in % non-aggregation when the pH was first raised to 11, then lowered to 3.
  • samples were sterilized using 0.2 ⁇ m PES filters into 15 mL sterile tubes, and then aliquoted into 1.5 mL sterile cryo vials (polypropylene, Corning cat. no. 430659). These samples were placed in storage boxes in humidity-controlled incubators. Three sets of about 1.0 mL samples were placed in 30°C conditions, and one set was placed in 4O 0 C. The remaining sample was stored at 4°C until all samples were analyzed by EF, IF, RALS. Remaining material was frozen for subsequent analysis by HPLC.
  • Acidic pH (3-5) resulted in increased aggregation, which increased with incubation over time at 30°C or 40 0 C.
  • alkaline pH (10 and 11) only incubation at 40 0 C resulted in aggregation.
  • the assays as described herein have been shown to be good indicators for AIC stability and are predicted to be good indicators for the structural stability of any conjugate molecule: RALS, IF, EF and SEC-HPLC. Based on the results of the above experiments, the following parameters were followed for design of compositions that provide for structurally stable conjugate molecules. The study will be designed to test components with pK values between 7 and 9. No salt will be used (the ionic strength study did suggest that low concentrations may be viable for adjusting osmolarity in the final formulation, since 10 mM NaCl did not substantially impact the molecule's stability).
  • Tables 10, 11 and 12 were prepared based on preparation of a decision matrix for evaluation of components as tested by RALS, EF, IF and HPLC-SEC.
  • the effects of time and temperature on AIC in various compositions was analyzed by fluorimetric assays and the results were scored by qualitative and comparative analysis in one of two ways. In some studies, compositions were numbered from best to worst (such as, 1 to 10, with 1 being the best) as judged by each assay at each time point. Each assay was scored for initial values, for changes in an acute temperature ramp, and for changes over time when incubated at 40 degrees C (to (0 time) vs t 7 (7 days), tj 4 (14 days) and t 28 (28 days); smaller changes were given higher scores).
  • the number was then converted to a score from 0 to 100, with 100 being the best. Other studies used the actual value from each assay rather then ranking the compositions. All these scores were averaged and reported in Tables 10, 11, 12.
  • the HPLC results were scored by mathematically spreading the actual % non- aggregation and Recovery values for each incubation time point, that is 0, 7 days, 14 days and 28 days, over a range from 0-100. The results from all three wavelengths were averaged for the score. The t 7> t ]4 , and t 28 HPLC data were assessed separately in order to give a greater weight to these data.
  • the thermal stress studies on AIC indicated that 40 degrees C would be useful in accelerated structural stability studies, as at this temperature the AIC is 10 degrees C below its thermal unfolding transition of about 50 to about 60 degrees C (as assessed by IF and EF studies).
  • RALS experiments demonstrate that AIC aggregates and precipitates at about 75 to about 80 degrees C.
  • the temperature appropriate for accelerated structural stability studies on other conjugate molecules can be determined by measuring the thermal unfolding transition temperature of the conjugate molecule to be analyzed and maintaining the temperature of the studies below about 10 degrees less than the unfolding transition temperature. Such determinations can be made by methods known by the skilled artisan and by IF and EF methods disclosed herein. IF and EF of AIC was examined with and without an acute temperature ramp (30 minutes, from 20 degrees C to 90 degrees C).
  • Sterile buffer was aliquoted and stored at 4 degrees C for use as controls in AIC determination and for injection on HPLC. After dialysis, the samples were filter-sterilized using 0.2 ⁇ m PES filters in a biosafety hood and aliquoted into 4 sterile cryovials. Three sets of each composition were prepared at 30 ⁇ g.mL and placed in a 40 degree C incubator. One set was used immediately for to studies. Control composition is sodium dibasic phosphate 2OmM at pH 7.2 (JT Baker); Benzoate (Ben) is sodium benzoate 2OmM
  • Deg Degradation products formed; *10mm Sodium Phosphate, 141.7 mM NaCl, pH 7.2
  • Histidine at pH 7.5; Histidine at pH 8.0; Phosphate at pH 7.5, pH 8.0, pH 7.0; and Histidine at pH 7.0 and pH 6.5 are selected for use in compositions.
  • Histidine at pH 7.5 is used in compositions comprising a conjugate molecule.
  • the affects of various amino acid stabilizers on AIC were assessed. The set selected below were chosen based on their use as injectable parenteral agents in license drug products.
  • the compositions comprising amino acids to be tested were prepared for dialysis with 2OmM Histidine and 5OmM of the amino acid and pH was adjusted to 7.5. The compositions were filtered using 0.2 ⁇ m Nalgene filters. As controls, the compositions containing Histidine alone were also prepared at 5mM, 2OmM and
  • the six amino acids selected had similar stabilizing effects. They are: 5mM Histidine, Glycine, Isoleucine, Leucine, Proline, and Alanine. For combination studies, Glycine was the primary selected Amino Acid.
  • compositions containing combinations including of 2 buffers, 2 Amino Acids, and 2 Carbohydrates. These Components were chosen for the study based on data obtained through 28 days of stability studies, except Carbohydrate, which was chosen from data through 14 days of studies. Compositions were designed to simplify comparison between changes in single Components while reducing the number of compositions required for assessment. Compositions were prepared with the Components listed in Table 13 (pH 7. 5) using the combinations shown and the procedures as described herein. One set was used immediately for t 0 studies.
  • compositions were analyzed for osmolarity after final dilution and filtering (to compositions).
  • AlC concentrations and recoveries at each time point were determined using absorbance at 280 nM, and are presented in Table 14.
  • compositions were further analyzed by RALS, IF, EF and SEC-HPLC by methods as described herein.
  • Table 14 Decision Table for Selecting the Components for Composition
  • compositions 3, 4, 6, and 8 were selected (all had similar results).
  • Compositions 1 and 2 are control compositions and do not have sufficient osmolarity to be chosen as the final composition.
  • compositions 4-6 caused a decrease in stability. Inclusion of Mannose (Composition 9) in the place of Sucrose (Composition 4) resulted in a composition causing a considerable drop in stability. Adding Isoleucine (Composition 10) and reducing Sucrose (Composition 4) concentration resulted in a drop in stability. Phosphate buffered compositions (11-14) did not perform as well as Histidine buffered compositions overall. However, in the HPLC data alone, the Phosphate buffered compositions performed almost as well as the Histidine buffered compositions.
  • Study 2 Preparation of Compositions for Combination Study 2 To further investigate the effect of combining components identified as good stabilizers, the study presented here assessed compositions containing 1 buffer, 2 Amino Acids, and 3 Carbohydrates as stabilizers. They were chosen for the study based on data obtained through 28 days of stability studies. Compositions were designed to explore the use of Sorbitol since the data from the 28-day incubation of AIC indicated that Sorbitol stabilizes AIC better than Sucrose.
  • compositions were prepared with the components listed in Table 15. The pH of each composition was adjusted to 7.5, and then each composition was prepared as described herein.
  • the AIC concentrations and recoveries after dialysis and filtration (t 0 ), and after 7, 14 and 29-day incubation at 40°C were determined using absorbance at 280 nm.
  • compositions were further analyzed by RALS, IF, EF and SEC-HPLC by methods as described herein.
  • Table 16 To select composition, a decision matrix table, Table 16 was constructed. Table 16
  • compositions 3, 7, 9 and 10 were selected. The four all had similar results
  • composition 10 was selected for use. Increasing Sorbitol concentration increased stability as demonstrated with Composition 2 vs. 3 and Composition 6 vs. 7. Replacing Leucine (Composition 9) with Glycine (Composition 7) caused a slight decrease in stability. Composition 3, which contained Sorbitol without Glycine or Leucine, proved to be a better stabilizer than compositions using Sorbitol combined with Glycine or Leucine.
  • compositions containing Histidine rather than Phosphate exhibited higher stability based on their overall score (Compositions 5 and 6), although Phosphate combined with Sorbitol stabilized AIC almost as well as a composition containing Histidine and Sorbitol when considering HPLC data alone (Composition 3 vs. 5).
  • compositions were prepared with the Components listed in Table 17 using the combinations shown.
  • the pH of each composition was adjusted to 7.5, and the compositions prepared as described herein.
  • the composition was intended to be 57 mM NaCl.
  • compositions were further analyzed by RALS, IF, EF and SEC-HPLC by methods as described herein. Table 18 provides a matrix for selecting combinations of compositions.
  • compositions from the combination study were selected: 1, 3, 4, 5, 12. The compositions all had similar results.
  • compositions 6-11 Composition 1 scored better in this round of composition than it had in the previous round, but it does not have sufficient osmolality.
  • Compositions containing Leucine or Proline with Histidine and Sorbitol Compositions 3 and 4 provided better stabilization than compositions without Leucine or Proline (Composition 1).
  • Leucine and Proline are better components than Glycine when they are combined with Sorbitol (Composition 3 and 4 vs. 2), but Glycine is a better stabilizer without Sorbitol (Composition 5) than either Composition 3 or 4. Study 4
  • compositions were prepared with the Components listed in Table 19 using the combinations shown. The pH of each composition was adjusted to 7.5, and the compositions were prepared according to the method described herein.
  • compositions were prepared such that the use of Histidine versus Phosphate could be confirmed.
  • the AIC concentrations and recoveries after dialysis and after filtration (to) after all time points were determined using absorbance at 280 run, and are shown in Table 20.
  • compositions with Combined Components AIC Concentration of AIC at A 2 so
  • compositions were further analyzed by RALS, IF, EF and SEC-HPLC by methods as described herein.
  • the results of RAL measurements are shown in Figure 5.
  • compositions containing Histidine (1 and 3) provided better stability than those containing Phosphate (2).
  • a composition with low Histidine concentration and high Glycine concentration (Composition 3) provided better stability than a composition with high Histidine concentration and a low Glycine concentration (Composition 1).
  • the data suggest that AIC is most stable in the absence of NaCl.
  • the best pH for AIC is 7.5 (range 7-9).
  • AIC is not substantially shear sensitive. Temperatures above 40°C result in degraded AIC. Temperature and pH stresses both produce insoluble and aggregated species.
  • the best component for maintaining pH for an AIC composition is Histidine at pH 7.5, followed by Histidine at pH 8.0, and Phosphate at pH 7.5 and pH 8.0.
  • the most desirable Amino Acids for a stable AIC composition are: 5mM Histidine alone, Glycine, Isoleucine, Leucine, Proline, and Alanine.
  • the best Carbohydrates for a stable AIC composition are: Lactose, Sucrose, and Mannose and Maltose. Maltose and Lactose are reducing sugars. Looking at HPLC results alone, Sorbitol and Glucose provided improved stability over all carbohydrates except the reducing sugars.
  • compositions can be used for liquid composition of structurally stable AIC at temperatures between about 2° degrees and about 8° degrees C: 5 mM Histidine, 285 mM Glycine; 20 mM Histidine, 270 mM Glycine; and 20 mM Histidine, 50 mM Glycine, and 3.8 % Sorbitol.

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Abstract

L'invention concerne des compositions contenant des molécules conjuguées structurellement stables à une température comprise entre environ 2 et 8 degrés C. dans certains modes de réalisation, la molécule conjuguée contient un antigène, tel qu'un allergène. Dans d'autres modes de réalisation, une molécule conjuguée contient un antigène Amb d'herbe à poux à 1°. Cette invention concerne également des procédé destinés à fabriquer et à utiliser ces compositions, ainsi que des procédés de modulation d'une réponse immunitaire chez un individu qui consiste à administrer une composition contenant une molécule conjuguée structurellement stable telle que décrite dans cette invention.
PCT/US2006/007571 2005-03-04 2006-03-03 Compositions contenant des molecules conjuguees structurellement stables WO2006096497A2 (fr)

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EP06736828A EP2007421A2 (fr) 2005-03-04 2006-03-03 Vaccins comprenant des oligonucleotides immunostimulateurs (iss), desquels iss sont conjugues a des antigenes et stabilises dans des conditions de tampon et par d'autres excipients
JP2007558260A JP2008531722A (ja) 2005-03-04 2006-03-03 構造的に安定なコンジュゲート分子を含む組成物
CA002600036A CA2600036A1 (fr) 2005-03-04 2006-03-03 Compositions contenant des molecules conjuguees structurellement stables
NZ561144A NZ561144A (en) 2005-03-04 2006-03-03 Vaccines comprising oligonucleotides having immunostimulatory sequences (ISS) wherein the ISS are conjugated to antigens and stabilized by buffer conditions and further excipients
AU2006220835A AU2006220835B2 (en) 2005-03-04 2006-03-03 Vaccines comprising oligonucleotides having immunostimulatory sequences (ISS) wherein the ISS are conjugated to antigens and stabilized by buffer conditions and further excipients

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KR20070110901A (ko) 2007-11-20
NZ561144A (en) 2009-09-25
US7718622B2 (en) 2010-05-18
EP2007421A2 (fr) 2008-12-31
JP2008531722A (ja) 2008-08-14
CA2600036A1 (fr) 2006-09-14
AU2006220835B2 (en) 2012-01-19
USRE45196E1 (en) 2014-10-14
WO2006096497A3 (fr) 2007-05-03
US20070036807A1 (en) 2007-02-15
SG160336A1 (en) 2010-04-29
CN101171033A (zh) 2008-04-30
AU2006220835A1 (en) 2006-09-14

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